ABSTRACT Songbird research has made many seminal contributions to the fields of ethology, endocrinology, physiology, ecology, evolution and neurobiology. Genome manipulation is thus a promising new methodological strategy to enhance the existing strengths of the songbird system to advance and expand fundamental knowledge of how genetic sequences and regulation of genomic function support complex natural learned behaviors. In zebra finches ( Taeniopygia guttata ) in particular, a rich set of questions about the complex process of developmental song learning in juvenile males has been defined. This Review uses one area of zebra finch song learning to demonstrate how genome editing can advance causal investigations into known genome–brain–behavior relationships. Given the number and diversity of songbird species, comparative work leveraging genome manipulation would expand the influence of these birds in additional fields of ecology and evolution for song learning and other behaviors.

ABSTRACT We hypothesised that daily food availability times serve as an ‘epigenetic’ factor and affect reproductive physiology in continuously reproducing species. This we tested by measuring mRNA expression of genes coding for enzymes involved in DNA methylation–demethylation ( dnmt , tet ) and histone modification ( hat1 , hdac ) in the hypothalamus, liver and gonads of male and female zebra finches that were paired for a year under 12 h light:12 h dark conditions with food availability restricted to 4 h in the morning (morning FA group) or evening (evening FA group), with controls provided with food ad libitum . The overall hypothalamic and hepatic expression patterns of hat1 and hdac were similar but those of dnmt and tet were different between males and females. Irrespective of the timing of food availability, both hat1 and hdac mRNA levels were increased in the hypothalamus, but not in the liver, in which hat1 mRNA levels were increased in the morning FA group. While hypothalamic tet levels were higher in evening FA males, hepatic tet levels were higher in morning FA birds ( tet1 , only males). Gonadal expression levels similarly varied and showed sex differences. Histone-modifying genes did not show food availability effects, except for elevated testicular hdac3 levels. Similarly, testicular dnmt3b and tet2 mRNA levels were increased and decreased in morning and evening FA groups, respectively, whereas ovarian dnmt1 and tet2 levels were reduced in morning FA and tet1 levels were reduced in evening FA groups. The present results suggest that an enforced daily feeding schedule in the long term could serve as a conditioning environment that shapes overall hypothalamic regulation, and liver and gonadal function at the epigenetic level in diurnal vertebrates.

ABSTRACT The acute effects of an energy-intensive activity such as exercise may alter an animal's redox homeostasis, although these short-term effects may be ameliorated by chronic exposure to that activity, or training, over time. Although well documented in mammals, how energy-intensive training affects the antioxidant system and damage by reactive species has not been investigated fully in flight-trained birds. We examined changes to redox homeostasis in zebra finches exposed to energy-intensive activity (60 min of perch-to-perch flights twice a day), and how exercise training over many weeks affected this response. We measured multiple components of the antioxidant system: an enzymatic antioxidant (glutathione peroxidase, GPx) and non-enzymatic antioxidants (measured by the OXY-adsorbent test) as well as a measure of oxidative damage (d-ROMs). At no point during the experiment did oxidative damage change. We discovered that exposure to energy-intensive exercise training did not alter baseline levels of GPx, but induced exercise-trained birds to maintain a higher non-enzymatic antioxidant status as compared with untrained birds. GPx activity was elevated above baseline in trained birds immediately after completion of the second 1 h flight on each of the three sampling days, and non-enzymatic antioxidants were acutely depleted during flight after 13 and 44 days of training. The primary effect of exercise training on the acute response of the antioxidant system to 2 h flights was increased coordination between the enzymatic (GPx) and non-enzymatic components of the antioxidant system of birds that reduced oxidative damage associated with exercise.

ABSTRACT Understanding the regulation of social behavioural expression requires insight into motivational and performance aspects. While a number of studies have independently assessed these aspects of social behaviours, few have examined how they relate to each other. By comparing behavioural variation in response to live or video presentations of conspecific females, we analysed how variation in the motivation to produce courtship song covaries with variation in performance aspects of courtship song in male zebra finches ( Taeniopygia guttata ). In agreement with previous reports, we observed that male zebra finches were less motivated to produce courtship songs to videos of females than to live presentations of females. However, we found that acoustic features that reflect song performance were not significantly different between songs produced in response to videos of females, and those produced in response to live females. For example, songs directed at video presentations of females were just as fast and stereotyped as songs directed at live females. These experimental manipulations and correlational analyses reveal a dissociation between motivational and performance aspects of birdsong and suggest a refinement of neural models of song production and control. In addition, they support the efficacy of videos to study both motivational and performance aspects of social behaviours.

ABSTRACT The song of the adult male zebra finch is a well-studied example of a learned motor sequence. Song bouts begin with a variable number of introductory notes (INs) before actual song production. Previous studies have shown that INs progress from a variable initial state to a stereotyped final state before each song. This progression is thought to represent motor preparation, but the underlying mechanisms remain poorly understood. Here, we assessed the role of sensory feedback in the progression of INs to song. We found that the mean number of INs before song and the progression of INs to song were not affected by removal of two sensory feedback pathways (auditory or proprioceptive). In both feedback-intact and feedback-deprived birds, the presence of calls (other non-song vocalizations), just before the first IN, was correlated with fewer INs before song and an initial state closer to song. Finally, the initial IN state correlated with the time to song initiation. Overall, these results show that INs do not require real-time sensory feedback for progression to song. Rather, our results suggest that changes in IN features and their transition to song are controlled by internal neural processes, possibly involved in getting the brain ready to initiate a learned movement sequence.

ABSTRACT Recent studies of the onset of breeding in long-day photoperiodic breeders have focused on the roles of type 2 and 3 iodothyronine deiodinases (DIO2 and DIO3) in the conversion of thyroxine (T4) to triiodothyronine (T3) and subsequent activation of the reproductive axis. It has been hypothesized that an increase in DIO2 and a reciprocal decrease in DIO3 causes the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus, setting off a reproductive cascade, and that this DIO mechanism for GnRH release is conserved across vertebrate taxa. We sought to test whether social cues that are known to stimulate reproductive behaviors can activate the DIO system to initiate reproduction in a non-photoperiodic bird, the zebra finch ( Taeniopygia guttata ). Isolation of males and subsequent presentation of females did not increase DIO2 or GnRH expression in the hypothalamus, nor did it decrease gonadotropin-inhibitory hormone (GnIH) or DIO3. Males receiving a female stimulus showed significantly higher mRNA expression and immunoreactive cell count of the immediate-early gene early growth response protein 1 (EGR-1) than isolated males, indicating hypothalamic activation in response to a female. Cells immunoreactive for EGR-1 were not co-localized with those immunoreactive for GnRH. Reproductive behaviors (singing, copulation attempts and overall activity) were significantly higher in males receiving a female stimulus. This study presents a social effect on behavior and EGR-1 expression in the hypothalamus of males in response to females, but more research is needed to determine whether the DIO2 system and the GnRH system are responsive to social stimulation in this species.

ABSTRACT Although fever (a closely regulated increase in body temperature in response to infection) typically is beneficial, it is energetically costly and may induce detrimentally high body temperatures. This can increase the susceptibility to energetic bottlenecks and risks of overheating in some organisms. Accordingly, it could be particularly interesting to study fever in small birds, which have comparatively high metabolic rates and high, variable body temperatures. We therefore investigated two aspects of fever and other sickness behaviours (circadian variation, dose dependence) in a small songbird, the zebra finch. We injected lipopolysaccharide (LPS) at the beginning of either the day or the night, and subsequently monitored body temperature, body mass change and food intake for the duration of the response. We found pronounced circadian variation in the body temperature response to LPS injection, manifested by (dose-dependent) hypothermia during the day but fever at night. This resulted in body temperature during the peak response being relatively similar during the day and night. Day-to-night differences might be explained in the context of circadian variation in body temperature: songbirds have a high daytime body temperature that is augmented by substantial heat production peaks during activity. This might require a trade-off between the benefit of fever and the risk of overheating. In contrast, at night, when body temperature is typically lower and less variable, fever can be used to mitigate infection. We suggest that the change in body temperature during infection in small songbirds is context dependent and regulated to promote survival according to individual demands at the time of infection.

ABSTRACT Spaced practice affects learning efficiency in humans and other animals. However, it is not well understood how spaced practice contributes to learning during development. Here, we show the behavioral significance of singing frequency in song development in a songbird, the zebra finch. Songbirds learn a complex song pattern by trial-and-error vocalizations as self-motivated practice, which is executed over a thousand times per day during the sensitive period of vocal learning. Notably, juveniles generate songs with a high frequency of singing in clusters with dense singing, whereas adults sing with low frequency in short clusters. This juvenile-specific clustered singing was characterized by clear separations of daily time for intense practice and rest. During the epochs of vocal practice in juveniles, the song structure approached that of song produced at the end of the day. In contrast, during the epochs of vocal rest, the structure of juvenile songs regressed toward that of songs produced at the beginning of the day, indicating a dynamic progression and regression of song development over the course of the day. When the singing frequency was manipulated to decrease it at the juvenile stage, the oscillation rate of song development was dramatically reduced. Although the juvenile-specific clustered singing occurred in non-tutored socially isolated birds or those with auditory deprivation, the diurnal oscillation of vocal development was only observed in non-tutored isolated juveniles. These results show the impact of ‘self-motivated’ vocal practice on diurnal song developmental plasticity, modulated by the amount of vocal output and auditory feedback.

Transitions to and from the air are critical for aerial locomotion and likely shaped the evolution of flying animals. Research on take-off demonstrates that legs generate greater body accelerations compared with wings, and thereby contribute more to initial flight velocity. Here, we explored coordination between wings and legs in two species with different wingbeat styles, and quantified force production of these modules during the final phase of landing. We used the same birds that we had previously studied during take-off: zebra finch ( Taeniopygia guttata, N =4) and diamond dove ( Geopelia cuneata, N =3). We measured kinematics using high-speed video, aerodynamics using particle image velocimetry, and ground-reaction forces using a perch mounted on a force plate. In contrast with the first three wingbeats of take-off, the final four wingbeats during landing featured ~2 times greater force production. Thus, wings contribute proportionally more to changes in velocity during the last phase of landing compared with the initial phase of take-off. The two species touched down at the same velocity (~1 m s −1 ), but they exhibited significant differences in the timing of their final wingbeat relative to touchdown. The ratio of average wing force to peak leg force was greater in diamond doves than in zebra finches. Peak ground reaction forces during landing were ~50% of those during take-off, consistent with the birds being motivated to control landing. Likewise, estimations of mechanical energy flux for both species indicate that wings produce 3–10 times more mechanical work within the final wingbeats of flight compared with the kinetic energy of the body absorbed by legs during ground contact.

SUMMARY Humans and songbirds are among the rare animal groups that exhibit socially learned vocalizations: speech and song, respectively. These vocal-learning capacities share a reliance on audition and cortico-basal ganglia circuitry, as well as neurogenetic mechanisms. Notably, the transcription factors Forkhead box proteins 1 and 2 (FoxP1, FoxP2) exhibit similar expression patterns in the cortex and basal ganglia of humans and the zebra finch species of songbird, among other brain regions. Mutations in either gene are associated with language disorders in humans. Experimental knock-down of FoxP2 in the basal ganglia song control region Area X during song development leads to imprecise copying of tutor songs. Moreover, FoxP2 levels decrease naturally within Area X when zebra finches sing. Here, we examined neural expression patterns of FoxP1 and FoxP2 mRNA in adult Bengalese finches, a songbird species whose songs exhibit greater sequence complexity and increased reliance on audition for maintaining their quality. We found that FoxP1 and FoxP2 expression in Bengalese finches is similar to that in zebra finches, including strong mRNA signals for both factors in multiple song control nuclei and enhancement of FoxP1 in these regions relative to surrounding brain tissue. As with zebra finches, when Bengalese finches sing, FoxP2 is behaviorally downregulated within basal ganglia Area X over a similar time course, and expression negatively correlates with the amount of singing. This study confirms that in multiple songbird species, FoxP1 expression highlights song control regions, and regulation of FoxP2 is associated with motor control of song.

SUMMARY Take-off mechanics are fundamental to the ecology and evolution of flying animals. Recent research has revealed that initial take-off velocity in birds is driven mostly by hindlimb forces. However, the contribution of the wings during the transition to air is unknown. To investigate this transition, we integrated measurements of both leg and wing forces during take-off and the first three wingbeats in zebra finch ( Taeniopygia guttata , body mass 15 g, N =7) and diamond dove ( Geopelia cuneata , body mass 50 g, N =3). We measured ground reaction forces produced by the hindlimbs using a perch mounted on a force plate, whole-body and wing kinematics using high-speed video, and aerodynamic forces using particle image velocimetry (PIV). Take-off performance was generally similar between species. When birds were perched, an acceleration peak produced by the legs contributed to 85±1% of the whole-body resultant acceleration in finch and 77±6% in dove. At lift-off, coincident with the start of the first downstroke, the percentage of hindlimb contribution to initial flight velocity was 93.6±0.6% in finch and 95.2±0.4% in dove. In finch, the first wingbeat produced 57.9±3.4% of the lift created during subsequent wingbeats compared with 62.5±2.2% in dove. Advance ratios were <0.5 in both species, even when taking self-convection of shed vortices into account, so it was likely that wing–wake interactions dominated aerodynamics during wingbeats 2 and 3. These results underscore the relatively low contribution of the wings to initial take-off, and reveal a novel transitional role for the first wingbeat in terms of force production.

SUMMARY Trade-offs between self-maintenance processes can affect life-history evolution. Integument replacement and the stress response both promote self-maintenance and affect survival in vertebrates. Relationships between the two processes have been studied most extensively in birds, where hormonal stress suppression is down regulated during molt in seasonal species, suggesting a resource-based trade-off between the two processes. The only species found to differ are the rock dove and Eurasian tree sparrow, at least one of which performs a very slow molt that may reduce resource demands during feather growth, permitting investment in the stress response. To test for the presence of a molt–stress response trade-off, we measured hormonal stress responsiveness during and outside molt in two additional species with extended molts, red crossbills ( Loxia curvirostra ) and zebra finches ( Taeniopygia guttata ). We found that both species maintain hormonal stress responsiveness during molt. Further, a comparative analysis of all available species revealed a strong relationship between molt duration and degree of hormonal suppression. Though our results support trade-off hypotheses, these data can also be explained by alternative hypotheses that have not been formally addressed in the literature. We found a strong relationship between stress suppression and seasonality of breeding and evidence suggesting that the degree of suppression may be either locally adaptable or plastic and responsive to local environmental conditions. We hypothesize that environmental unpredictability favors extended molt duration, which in turn allows for maintenance of the hormonal stress response, and discuss implications of a possible trade-off for the evolution of molt schedules.

SUMMARY Coordination of different motor systems for sound production involves the use of feedback mechanisms. Song production in oscines is a well-established animal model for studying learned vocal behavior. Whereas the online use of auditory feedback has been studied in the songbird model, very little is known about the role of other feedback mechanisms. Auditory feedback is required for the maintenance of stereotyped adult song. In addition, the use of somatosensory feedback to maintain pressure during song has been demonstrated with experimentally induced fluctuations in air sac pressure. Feedback information mediating this response is thought to be routed to the central nervous system via afferent fibers of the vagus nerve. Here, we tested the effects of unilateral vagotomy on the peripheral motor patterns of song production and the acoustic features. Unilateral vagotomy caused a variety of disruptions and alterations to the respiratory pattern of song, some of which affected the acoustic structure of vocalizations. These changes were most pronounced a few days after nerve resection and varied between individuals. In the most extreme cases, the motor gestures of respiration were so severely disrupted that individual song syllables or the song motif were atypically terminated. Acoustic changes also suggest altered use of the two sound generators and upper vocal tract filtering, indicating that the disruption of vagal feedback caused changes to the motor program of all motor systems involved in song production and modification. This evidence for the use of vagal feedback by the song system with disruption of song during the first days after nerve cut provides a contrast to the longer-term effects of auditory feedback disruption. It suggests a significant role for somatosensory feedback that differs from that of auditory feedback.

SUMMARY It can be hypothesized that species with a wide or variable food niche are able to adjust their digestive physiology to current food type. In diet specialists, however, the capacity for such presumably costly plasticity is not necessary and flexibility of digestive physiology should be lower. Recently, we found that ontogenetic changes in the activity of digestive enzymes in house sparrow, a species that gradually consumes more carbohydrates during ontogeny, are strongly modified by diet composition. In the present study we examined digestive flexibility of nestling and adult zebra finches, typical diet specialists that consume only seeds after hatching. Both adult and nestling zebra finches could not thrive on a protein-rich and carbohydrate-free diet that supported normal development of young house sparrows. Mass-specific activity of intestinal carbohydrases (maltase and sucrase) was not elevated by higher diet carbohydrate content in both nestling and adult birds. Mass-specific activity of maltase changed less during ontogenetic development in zebra finch than in house sparrow. We conclude that the digestive physiology of zebra finch is adapted to process carbohydrate-rich food after hatching and is much less flexible than in house sparrow. We hypothesize that this difference might reflect the lack of a diet switch during ontogeny or result from high specialization to a narrow diet niche.

SUMMARY The vitellogenin/very low density lipoprotein receptor (VTG/VLDL-R), a 95 kDa protein that belongs to the low density lipoprotein receptor gene family,mediates the uptake of yolk precursors by developing follicles during oocyte growth. However, the extent to which variation in VTG/VLDL-R expression plays a role in determining inter-individual variation in reproductive phenotype(e.g. follicle or egg size) is not known. Here we show that the mRNA sequence of the zebra finch ( Taeniopygia guttata ) VTG/VLDL-R shows a high degree of sequence identity (92%) with chicken VTG/VLDL-R mRNA. Using quantitative real-time PCR we measured transcriptional expression of VTG/VLDL-R mRNA in various tissues, and for different stages of oocyte growth,in individual female zebra finches. VTG/VLDL-R mRNA was expressed at high levels in vitellogenic oocytes and in skeletal muscle, and was also detectable in liver, but these tissues expressed different splice variants: the short-form LR8–in oocytes and liver, and the LR8+ form in skeletal muscle. There was significant temporal variation in VTG/VLDL-R expression during follicle growth, with highest levels in ovary and a gradual decrease from pre-F3 to F1 vitellogenic follicles. Variation in ovary mRNA expression was correlated with inter-individual variation in clutch size and laying interval. Furthermore, variation in F3 follicle VTG/VLDL-R mRNA expression was correlated with inter-individual variation in egg mass and F1 follicle mass,suggesting that VTG/VLDL receptor mRNA expression is a key determinant of inter-individual variation in reproductive phenotype.

SUMMARY Reductions in hematological parameters among laying birds are well reported, but the cause of this anemia is not known. We tested specific predictions generated from several, non-mutually exclusive hypotheses for mechanisms underlying reproductive anemia associated with egg production(hemodilution, transient suppression of erythropoiesis, resource dependence)in relation to (1) the time-course of development and recovery from anemia,(2) changes in specific hematological traits, and (3) the effect of diet quality, in female zebra finches ( Taeniopygia guttata ). Female zebra finches showed marked decreases in hematocrit (∼6%), red blood cell counts(∼8%), and plasma hemoglobin concentration (∼9%) during egg production, even on a high-quality ad libitum diet, consistent with an effect of hemodilution associated with yolk precursor production. However,our results provide strong support for the hypothesis that erythropoiesis is transiently suppressed during egg-laying and that the recovery from anemia is relatively long-lasting, extending through incubation and hatching periods. Decreased hematocrit, red blood cell counts, and hemoglobin concentration did not recover at clutch completion, but showed evidence of recovery to baseline pre-breeding levels at hatching. More importantly, there was significant time-dependent variation in the proportion of reticulocytes, which increased at clutch completion but peaked at hatching 10–12 days after clutch completion, and in mean red blood cell volume, which showed a significant increase at clutch completion; consistent with enhanced production and release of larger immature cells into the circulation following suppression of erythropoiesis. Finally, we found no evidence for resource dependence of anemia associated with egg production in relation to diet quality, i.e. exogenous lipid and protein resources available to the laying female. This study demonstrates that transient suppression of erythropoiesis and,subsequently, increased reticulocytosis, are key components of reproductive anemia in egg-laying females.

SUMMARY The `cost of reproduction' (i.e. the trade-off between current reproduction and future fecundity and/or survival) is a central concept in life history theory, yet we still know very little about the physiological mechanisms underlying such costs. Recently it has been recognized that reproduction itself or the regulatory (hormonal) mechanisms underlying reproduction might result in `costs' (cf. resource-allocation based mechanisms). As one example,it has been suggested that the decrease in hematocrit observed during egg production in birds might be due to antagonistic pleiotropic effects of estrogens. This could generate costs of reproduction by reducing oxygen-carrying capacity during subsequent aerobically demanding stages such as chick-provisioning. Here we show that the reduction in hematocrit during egg-laying is dependent on receptor-mediated actions of endogenous estrogens:blocking estrogen receptors using the anti-estrogen tamoxifen reduces the decrease in hematocrit during egg production in female zebra finches( Taeniopygia guttata ) such that hematocrit at the 1-egg stage is not significantly different than pre-breeding, baseline values. We also show that both pre-breeding hematocrit and the decrease in hematocrit associated with egg production are repeatable, and that females with the highest pre-breeding hematocrit values tend to show the largest decreases in hematocrit during egg production. We suggest that hematological changes during egg production are a good candidate mechanism for a regulatory-network based trade-off involving antagonistic pleiotropic effects of estrogens, which otherwise have essential reproductive functions.

SUMMARY The effects of environmental stress on the physiology and behaviour of higher vertebrates has become an important avenue of research in recent years. Evidence from recent studies has suggested that the avian stress-related hormone corticosterone (CORT) may play a role in immunocompetence and sexual selection. We tested whether CORT is immunosuppressive by studying humoral and cell-mediated immune responses in populations of captive zebra finches selected for divergent peak levels of CORT. We also investigated whether selection for peak CORT has an effect on the quality of several sexually selected regions of the male zebra finch; in addition we compared morphometric parameters and the dominance ranking in males from the different selection lines. We also tested whether different components of the immune system compete for limited resources. We found that selection for divergent levels of peak CORT had little effect on humoral immunity, male sexual signal quality or dominance ranking. However, contrary to expectations, we did find a positive relationship between CORT titre and cell-mediated immunity, as well as a greater cell-mediated response in the birds selected for high CORT titre than those selected for low CORT titre. Consistent with predictions, significant negative relationships were found between both testosterone and CORT titre on humoral immunity. Birds from the low CORT lines were significantly larger in terms of skeletal size than those from the high CORT lines. Overall, our results suggest that the cell-mediated immune response is associated with a reduction in the humoral response, but only in males, and that there is no simple relationship between peak CORT levels and immune function.

SUMMARY During avian egg production, oestrogen mediates marked increases in hepatic lipid production and changes in the diameter of assembled very-low density lipoprotein (VLDL). A nearly complete shift from generic VLDL (∼70 nm in diameter), which transports lipids to peripheral tissues, to yolk-targeted VLDL (VLDLy) (∼30 nm), which supplies the yolk with energy-rich lipid, has been observed in the plasma of laying domestic fowl. We validated an established dynamic laser scattering technique for a passerine songbird Taeniopygia guttata , the zebra finch, to characterize the dynamics of VLDL particle diameter distribution in relation to egg production. We predicted that non-gallinaceous avian species that have not been selected for maximum egg production would exhibit less dramatic shifts in lipid metabolism during egg production. As predicted, there was considerable overlap between the VLDL particle diameter distributions of laying and non-laying zebra finches. But unexpectedly, non-laying zebra finches had VLDL diameter distributions that peaked at small particles and had relatively few large VLDL particles. As a result, laying zebra finches, in comparison, had diameter distributions that were shifted towards larger VLDL particles. Nevertheless,laying zebra finches, like laying chickens, had larger proportions of particles within proposed VLDLy particle diameter ranges than non-laying zebra finches (e.g. sVLDLy: 50% vs 37%). Furthermore, zebra finches and chickens had similar modal (29.7 nm in both species) and median (32.7 nm vs 29.6 nm) VLDL particle diameters during egg production. Therefore,although zebra finches and chickens exhibited opposing directional shifts in VLDL particle diameter distribution during egg production, the modifications to VLDL particle structure in both species resulted in the realization of a common goal, i.e. to produce and maintain a large proportion of small VLDL particles of specific diameters that are capable of being incorporated into newly forming egg yolks.)

SUMMARY Carotenoid-based signals are thought to be indicators of male quality because they must be obtained from the diet and might thus indicate the ability of individuals to gather high-quality food. However, carotenoids are also known to have important physiological functions as immunoenhancers and antioxidants, and, as such, carotenoid-based sexual traits have also been suggested to reflect the health and antioxidant status of their bearers. This last idea is based on the hypothesis that carotenoids that are allocated to sexual signals are no longer available for the detoxification system. Recently, this hypothesis has been challenged on the grounds that the antioxidant activity is not the main biological role of carotenoids. Instead,carotenoid-based sexual traits might signal the availability of other non-pigmentary antioxidant molecules that might protect carotenoids from free radical attacks and make them available for sexual advertisements. We tested this hypothesis in the zebra finch, a passerine species with a carotenoid-based signal: the colour of the bill. We simultaneously manipulated the availability of carotenoids and of a non-pigmentary antioxidant(melatonin) in the drinking water. If the antioxidant properties of melatonin protect carotenoids from oxidation, we predict that birds supplemented with melatonin should have redder bills than birds not supplemented with melatonin,and that birds supplemented with carotenoids and melatonin should have redder bills than birds supplemented with carotenoids alone. Our findings are in agreement with these predictions since carotenoid and melatonin supplementation had an additive effect on bill colour. To our knowledge this is the first experimental evidence that a non-pigmentary antioxidant enhances the expression of a carotenoid-based sexual trait.