Seasonal Changes in Courtship Song and the Medial Preoptic Area in Male European Starlings (Sturnus vulgaris)

In male starlings (Sturnus vulgaris) courtship song plays a critical role in mate attraction. During the breeding season courtship song occurs prior to copulation and appears to reflect male sexual arousal. Outside the breeding season starlings sing, but song appears unrelated to reproduction. The aromatization of testosterone (T), likely within the medial preoptic nucleus (POM), is critical for the expression of male sexual arousal. The present study was performed to determine whether seasonal changes in the POM might relate to seasonal changes in courtship singing behavior in male starlings. T concentrations, the volume of the POM, and aromatase within the POM were examined both during and outside of the breeding season in male starlings. Song was also recorded at these times both with and without a female present. The POM was largest and contained dense aromatase immunostaining only during the spring breeding season, when T concentrations were highest and males responded to a female with an increase in courtship song. Outside the breeding season the volume of the POM was small, T concentrations were low, and males displayed no changes in song expression in response to female conspecifics. Song bout length was positively related to POM volume, and males sang longer songs in spring. Only males with nestboxes in spring responded to a female, and the POM tended to be larger in these males, suggesting that nestbox possession might influence neuroplasticity within the POM. Overall, the findings suggest that T-dependent plasticity and aromatase activity within the POM might regulate courtship singing in a wild songbird.     

Seasonal changes in courtship song and the medial preoptic area in male European starlings (Sturnus vulgaris)

In male starlings (Sturnus vulgaris) courtship song plays a critical role in mate attraction. During the breeding season courtship song occurs prior to copulation and appears to reflect male sexual arousal. Outside the breeding season starlings sing, but song appears unrelated to reproduction. The aromatization of testosterone (T), likely within the medial preoptic nucleus (POM), is critical for the expression of male sexual arousal. The present study was performed to determine whether seasonal changes in the POM might relate to seasonal changes in courtship singing behavior in male starlings. T concentrations, the volume of the POM, and aromatase within the POM were examined both during and outside of the breeding season in male starlings. Song was also recorded at these times both with and without a female present. The POM was largest and contained dense aromatase immunostaining only during the spring breeding season, when T concentrations were highest and males responded to a female with an increase in courtship song. Outside the breeding season the volume of the POM was small, T concentrations were low, and males displayed no changes in song expression in response to female conspecifics. Song bout length was positively related to POM volume, and males sang longer songs in spring. Only males with nestboxes in spring responded to a female, and the POM tended to be larger in these males, suggesting that nestbox possession might influence neuroplasticity within the POM. Overall, the findings suggest that T-dependent plasticity and aromatase activity within the POM might regulate courtship singing in a wild songbird.     

Lesions to the Medial Preoptic Area Affect Singing in the Male European Starling (Sturnus vulgaris)

The aromatization of testosterone (T) in the medial preoptic nucleus (POM) is known to regulate male courtship and sexual behaviors expressed prior to, and in anticipation of, copulation. Singing in male European starlings is used to attract mates prior to physical sexual contact, suggesting that the POM might be involved. The present study was performed to examine the effects of lesions targeting the POM on singing and courtship behavior in reproductively active male starlings. A significant decrease in song output and the gathering of green nest materials was observed in males with lesions to the POM compared to males with damage to brain areas outside of the POM. Lesions did not affect a male's tendency to remain near a female or to occupy a nestbox, suggesting that the effects of POM lesions were specific to courtship behaviors. Behavioral differences were not related to testis mass or volume, and GnRH immunoreactivity was observed within the hypothalamus and median eminence for each male, suggesting that the effects of POM lesions were related specifically to POM involvement in song expression rather than to a disruption of the GnRH axis. These results suggest a general role for the POM in the expression of behaviors related to sexual arousal or anticipation, including song.     

Immediate early gene activity in song control nuclei and brain areas regulating motivation relates positively to singing behavior during, but not outside of, a breeding context

In some species, such as songbirds, much is known about how the brain regulates vocal learning, production, and perception. What remains a mystery is what regulates the motivation to communicate. European starlings (Sturnus vulgaris) sing throughout most of the year, but the social and environmental factors that motivate singing behavior differ seasonally. Male song is highly sexually motivated during, but not outside of, the breeding season. Brain areas outside the song control system, such as the medial preoptic nucleus (POM) and ventral tegmental area (VTA), have been implicated in regulating sexually motivated behaviors in birds, including song. The present study was designed to explore whether these regions, as well as three song control nuclei [area X, the high vocal center (HVC), and the robust nucleus of the arcopallium (RA)], might be involved differentially in song produced within compared to outside of a breeding context. We recorded the behavioral responses of breeding and nonbreeding condition male starlings to the introduction of a female conspecific. Males did not show context-dependent differences in the overall amount of song sung. However, immunocytochemistry for the protein product of the immediate early gene cFOS revealed a positive linear relationship between the total amount of songs sung and number of cFOS-labeled cells in POM, VTA, HVC, and RA for birds singing during, but not outside of, a breeding context. These results suggest that these regions differentially regulate male song production depending on reproductive context. Overall the data support the hypothesis that the POM and VTA interact with the song control system, specifically HVC and RA, to regulate sexually motivated vocal communication in songbirds.     

Lesions to the medial preoptic area affect singing in the male European starling (Sturnus vulgaris)

The aromatization of testosterone (T) in the medial preoptic nucleus (POM) is known to regulate male courtship and sexual behaviors expressed prior to, and in anticipation of, copulation. Singing in male European starlings is used to attract mates prior to physical sexual contact, suggesting that the POM might be involved. The present study was performed to examine the effects of lesions targeting the POM on singing and courtship behavior in reproductively active male starlings. A significant decrease in song output and the gathering of green nest materials was observed in males with lesions to the POM compared to males with damage to brain areas outside of the POM. Lesions did not affect a male's tendency to remain near a female or to occupy a nestbox, suggesting that the effects of POM lesions were specific to courtship behaviors. Behavioral differences were not related to testis mass or volume, and GnRH immunoreactivity was observed within the hypothalamus and median eminence for each male, suggesting that the effects of POM lesions were related specifically to POM involvement in song expression rather than to a disruption of the GnRH axis. These results suggest a general role for the POM in the expression of behaviors related to sexual arousal or anticipation, including song.     

Antiphonal duetting and sex hormones in the tropical bush shrike Laniarius funebris (HARTLAUB).

We investigated changes in antiphonal duetting with phases of reproduction and circulating levels of luteinizing hormone, testosterone, and estradiol in slate-colored boubous (Laniarius funebris) breeding in aviaries. Frequency of overall male singing did not vary with reproductive phase while frequencies of female singing and female vocal responses to male song were reduced during incubation and feeding of nestlings. This resulted in significant changes in frequency of duetting. Males sang the sexual song type M1 more often during courtship and nest building than during the nestlings phase. Their territorial song types M2 and M4 did not vary with breeding phase. Females were less responsive to M1 during incubation and to M2 during the nest building and nestlings than during the courtship phase. Plasma levels of luteinizing hormone and testosterone increased in males from the prebreeding to the courtship phase. While testosterone decreased already during nest building and remained low during subsequent phases of reproduction, luteinizing hormone decreased during incubation and feeding of nestlings. Female luteinizing hormone levels were highest during nest building. Female estradiol levels decreased from nest building to incubation and increased again during subsequent nest building. Female testosterone levels were low but not basal and did not vary with phase. Neither the overall male and female singing frequencies nor the frequencies of male song types were correlated with hormonal state. However, female participation in territorial duets M4 correlated positively with their testosterone levels. It is suggested that in this monogamous, duetting species with prolonged pairbonds behavioral cues between the mates are more important than the hormonal state in control of male and female singing.     

Reward Associated with Singing Behavior Correlates with Opioid-Related Gene Expression in the Medial Preoptic Nucleus in Male European Starlings

Birdsong consists of species-specific learned vocal sequences that are used primarily to attract mates and to repel competitors during the breeding season. However, many birds continue to sing at times when vocal production has no immediate or obvious impact on conspecific behavior. The mechanisms that ensure that animals produce important behaviors in contexts in which the function of these behaviors is not immediate or obvious are not known. One possibility is that animals engage in such behaviors because they are associated with pleasure. Here we examined the hypothesis that male European starlings sing outside of the breeding season in part because the act of singing in this context is facilitated and/or maintained by opioid-mediated reward. We measured song-associated reward using a conditioned place preference (CPP) test in male starlings producing fall, non-breeding season-typical song. We used quantitative real time PCR to measure expression of the enkephalin opioid precursor preproenkephalin (PENK) and mu opioid receptors (MOR) in the medial preoptic nucleus (POM; a region in which opioids are implicated in both reward and starling fall song) and additionally the song control region HVC as a control. Starlings developed a strong preference for a place that had been paired previously with the act of producing fall-typical song, indicating that fall song production was associated with a positive affective state. Both PENK and MOR mRNA expression in the POM, but not HVC, correlated positively with both individual reward state (as reflected in CPP) and undirected singing behavior. These results suggest that singing induces opioid receptor and enkephalin expression in the POM and consequent reward, and/or that opioid release in the POM induced by individual or environmental factors (e.g., the presence of food, safety of a flock or the absence of predators) induces a positive affective state which then facilitates singing behavior.     

Testicular masculinization of vocal behavior in juvenile female Xenopus laevis reveals sensitive periods for song duration,rate, and frequency spectra

Summary In Xenopus laevis, adult males but not females produce courtship songs comprised of rapid trills. Two experiments were conducted to determine whether male-typical singing could be induced in females. At 6 different juvenile stages, male and female frogs were gonadectomized and implanted with testes, grown to sexual maturity, and tested for vocal behavior. All frogs with functional testicular implants sang; females sang as much as males. The frequency spectra of the clicks within trills were fully masculinized in females implanted at PM0, PM1, and PM2. There were deficiencies in song quality in females implanted late in juvenile life. Females receiving testis implants at PM3, PM4, and PM5 did not produce clicks with masculine spectral qualities. In a concurrent experiment, adult males and females were gonadectomized and implanted with testes or silicone tubes containing testosterone proprionate. When tested for vocal behavior 10 to 15 months after implantation, 8/10 androgen-treated males, 3/12 androgen-treated females, 5/5 testes-implanted males, and 2/4 testes-implanted females sang. The females that did sing spent much less time singing than males. The click rates of females were uniformly slower than males and no female produced clicks with a masculine frequency spectrum. Thus, testicular secretions can induce male-typical singing in females until late in juvenile development. However, females exhibit a progressive decline in vocal potential with increasing age, culminating in an almost complete loss of singing ability by adulthood.Abbreviations FFT fast Fourier transform - ICI inter-click interval - PM post-metamorphic - TP testosterone proprionate     

Differences in the social context of song production in captive male and female European starlings.

Studies on singing behavior in Oscine focus essentially on males and are carried out during the breeding season. Singing in females appears rare and is not well documented. However, females of several species can produce a complex song. Does this lack of data correspond to a real difference in males and females or to a non appropriate context of observation? We studied the vocal and social behavior of captive male and female European starlings during two periods: breeding and non-breeding periods. Our results indicated that females sang mostly in a non-breeding context: their singing behavior was strongly diminished when nestboxes were present in the aviary. Moreover, females sang more frequently when their closest neighbor was a female whereas males sang mostly when they had no immediate neighbor. These results indicate a difference between males and females for the context of song production.     

Social context affects testosterone-induced singing and the volume of song control nuclei in male canaries (Serinus canaria)

The contribution of social factors to seasonal plasticity in singing behavior and forebrain nuclei controlling song, and their interplay with gonadal steroid hormones are still poorly understood. In many songbird species, testosterone (T) enhances singing behavior but elevated plasma T concentrations are not absolutely required for singing to occur. Singing is generally produced either to defend a territory or to attract a mate and it is therefore not surprising that singing rate can be influenced by the sex and behavior of the social partner. We investigated, based on two independent experiments, the effect of the presence of a male or female partner on the rate of song produced by male canaries. In the first experiment, song rate was measured in dyads composed of one male and one female (M-F) or two males (M-M). Birds were implanted with T-filled Silastic capsules or with empty capsules as control. The number of complete song bouts produced by all males was recorded during 240 min on week 1, 2, 4, and 8 after implantation. On the day following each recording session, brains from approximately one-fourth of the birds were collected and the volumes of the song control nuclei HVC and RA were measured. T increased the singing rate and volume of HVC and RA but these effects were affected by the social context. Singing rates were higher in the M-M than in the M-F dyads. Also, in the M-M dyads a dominance-subordination relationship soon became established and dominant males sang at higher rates than subordinates in T-treated but not in control pairs. The differences in song production were not reflected in the size of the song control nuclei: HVC was larger in M-F than in M-M males and within the M-M dyads, no difference in HVC or RA size could be detected between dominant and subordinate males. At the individual level, the song rate with was positively correlated with RA and to a lower degree HVC volume, but this relationship was observed only in M-M dyads, specifically in dominant males. A second experiment, carried out with castrated males that were all treated with T and exposed either to another T-treated castrate or to an estradiol-implanted female, confirmed that song rate was higher in the M-M than in the M-F condition and that HVC volume was larger in heterosexual than in same-sex dyads. The effects of T on singing rate and on the volume of the song control nuclei are thus modulated by the social environment, including the presence/absence of a potential mate and dominance status among males.     

白腰文鸟发声行为的性别差异及其机制

通过声谱分析,研究了５－１２０日龄雌、雄白腰文鸟（Lonchura striata swinhoei)的声谱变化,及该时段３个主要发声控制核团）ＨＶＣ、ＲＡ、Ａrea X)体积、睾丸（睾酮）的相应改变。结果如下：①４５日龄以前,雌雄鸟只能发出简单鸣叫（call),鸣声基本不会鸣唱。②雄性ＨＶＣ,ＲＡ,ＡreaX体积均比雌性大２－６部。３个核团的大小发育不完全一致。各核团的快速生长期与鸣唱学习的主要时段（６０－１２０日龄）不同步,说明核团的个体发育可能不完全受发声行为的影响。③睾丸的充分发育（１２０日龄后）及血液中具有较高的睾酮水平是雄鸟发出成熟鸣唱语句的重要条件。     

Brief alteration in dopaminergic function during development causes deficits in adult reproductive behavior

Our previous research revealed dramatic increases in dopaminergic function in vocal control and auditory nuclei in male zebra finches during the period of song learning. Such increases were not seen in the hypothalamic areas examined. In the current study, we manipulated dopamine receptor function during this period to determine how this might affect later singing behavior. Males were implanted with osmotic minipumps providing 0, 0.5, or 5 microg/g/day of the mixed D1/D2 dopamine receptor antagonist cis-flupenthixol from day 45 until day 57. At approximately 86 days of age, males were given subcutaneous silastic implants containing a maintenance dose of androgen, in case antagonist treatment interfered with adult androgen secretion. One week later, they began a series of three weekly tests to determine if this early treatment affected courtship singing. Males treated with the low dose of cis-flupenthixol showed profound decrements in courtship singing and copulatory behavior. Unlike saline-treated controls, low-dose males sang to females infrequently. High-intensity courtship displays in which males dance towards females while singing were most affected. Despite their decreased courtship singing, low-dose males were interested in females. They approached females as frequently as males in the other two groups, but antagonist-treated males were less likely to follow females if they moved. Low-dose males also attempted to mount females significantly less often than high-dose males. High-dose males groomed significantly less frequently than males in the other two groups. Thus, brief early treatment with cis-flupenthixol had profound and long-lasting effects on female-directed singing and on copulatory behavior, despite androgen treatment.     

Social context affects testosterone‐induced singing and the volume of song control nuclei in male canaries (Serinus canaria)

The contribution of social factors to seasonal plasticity in singing behavior and forebrain nuclei controlling song, and their interplay with gonadal steroid hormones are still poorly understood. In many songbird species, testosterone (T) enhances singing behavior but elevated plasma T concentrations are not absolutely required for singing to occur. Singing is generally produced either to defend a territory or to attract a mate and it is therefore not surprising that singing rate can be influenced by the sex and behavior of the social partner. We investigated, based on two independent experiments, the effect of the presence of a male or female partner on the rate of song produced by male canaries. In the first experiment, song rate was measured in dyads composed of one male and one female (M‐F) or two males (M‐M). Birds were implanted with T‐filled Silastic capsules or with empty capsules as control. The number of complete song bouts produced by all males was recorded during 240 min on week 1, 2, 4, and 8 after implantation. On the day following each recording session, brains from approximately one‐fourth of the birds were collected and the volumes of the song control nuclei HVC and RA were measured. T increased the singing rate and volume of HVC and RA but these effects were affected by the social context. Singing rates were higher in the M‐M than in the M‐F dyads. Also, in the M‐M dyads a dominance‐subordination relationship soon became established and dominant males sang at higher rates than subordinates in T‐treated but not in control pairs. The differences in song production were not reflected in the size of the song control nuclei: HVC was larger in M‐F than in M‐M males and within the M‐M dyads, no difference in HVC or RA size could be detected between dominant and subordinate males. At the individual level, the song rate with was positively correlated with RA and to a lower degree HVC volume, but this relationship was observed only in M‐M dyads, specifically in dominant males. A second experiment, carried out with castrated males that were all treated with T and exposed either to another T‐treated castrate or to an estradiol‐implanted female, confirmed that song rate was higher in the M‐M than in the M‐F condition and that HVC volume was larger in heterosexual than in same‐sex dyads. The effects of T on singing rate and on the volume of the song control nuclei are thus modulated by the social environment, including the presence/absence of a potential mate and dominance status among males. 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Rufous‐capped Warblers Basileuterus rufifrons show seasonal,temporal and annual variation in song use

In the majority of songbird species, males have repertoires of multiple song types used for mate attraction and territory defence. The wood‐warblers (family Parulidae) are a diverse family of songbirds in which males of many migratory species use different song types or patterns of song delivery (known as ‘singing modes’) depending on context. The vocal behaviour of most tropical resident warblers remains undescribed, although these species differ ecologically and behaviourally from migratory species, and may therefore differ in their vocal behaviour. We test whether male Rufous‐capped Warblers Basileuterus rufifrons use distinct singing modes by examining song structure and context‐dependent variation in their songs. We recorded multiple song bouts from 50 male Warblers in a Costa Rican population over 3 years to describe seasonal, diel and annual variation in song structure and vocal behaviour. We found that Rufous‐capped Warbler songs are complex, with many syllable types shared both within and between males’ repertoires. Males varied their song output depending on context: they sang long songs at a high rate at dawn and during the breeding season, and shortened songs in the presence of a vocalizing female mate. Unlike many migratory species, Rufous‐capped Warblers do not appear to have different singing modes; they did not change the song variants used or the pattern of song delivery according to time of day, season or female vocal activity. Our research provides the first detailed vocal analysis of any Basileuterus warbler species, and enhances our understanding of the evolution of repertoire specialization in tropical resident songbirds.     

Androgen control of vocal control region volumes in a wild migratory songbird (Junco hyemalis) is region and possibly age dependent

Previous laboratory studies have shown that photoperiodic adult songbirds experience seasonal variations in singing frequency that correlate with plasma androgen levels, as well as changes in the brain regions that control singing (vocal control regions). The present study investigates naturally occurring seasonal changes in the sizes of these regions in a wild migratory species (dark-eyed junco, Junco hyemalis), with samples from adolescence to post-breeding fall migration. In adult males, the volumes of the vocal control regions area X and the higher vocal center (HVC) were large during the breeding season when birds were singing and androgen levels were high, and decreased in size after the breeding season when singing had stopped and androgen levels were low. HVC volume in adolescent males caught in the fall (no singing), when plasma androgen levels were low, was smaller than in breeding adults, thereby following the seasonal pattern of change in plasma androgen levels. In adolescent males, however, area X volume was the same as in breeding adults. Thus, area X size in adolescent male juncos may be testosterone independent. The seasonal pattern of robust nucleus of the archistriatum volume was similar to that of the HVC. The volumes of neither the magnocellular nucleus of the anterior neostriatum nor the nucleus rotundus, a control region, differed seasonally. Castration of breeding adult males caused both area X and HVC volumes to decrease compared to castrated controls with testosterone replacement, indicating that maintenance of these two region volumes is testosterone dependent in adults. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 391–402, 1997.     

Coordinated and dissociated effects of testosterone on singing behavior and song control nuclei in canaries (Serinus canaria)

Temperate zone songbirds that breed seasonally exhibit pronounced differences in reproductive behaviors including song inside and outside the breeding season. Springlike long daylengths are associated with increases in plasma testosterone (T) concentrations, as well as with increases in singing and in the volume of several brain nuclei known to control this behavior. The mechanisms whereby T can induce changes in behavior and brain, and whether or not these effects are differentially regulated, have recently begun to be examined, as has the question of the relative contributions of T and its androgenic and estrogenic metabolites to the regulation of this seasonal behavioral and neural plasticity. In this experiment, we examined the effects of T, 5alpha-dihydrotestosterone, or 17beta-estradiol treatment on castrated male canaries housed on short days and compared neural and behavioral effects in these males to similarly-housed males given only blank implants. We observed that only T treatment was effective in eliciting significant increases in singing behavior after 11 days of hormone exposure. In addition, T alone was effective in increasing the volume of a key song production nucleus, HVC. However, at this time, none of the steroids had any effects on the volumes of two other song control nuclei, Area X of the medial striatum and the robust nucleus of the arcopallium (RA), that are efferent targets of HVC, known to be regulated by androgen in canaries and also to play a role in the control of adult song. T can thus enhance singing well before concomitant androgen-induced changes in the song control system are complete.     

Song correlates with social context,testosterone and body condition in male barn swallows

Bird song, like many other male secondary sexual characters, may have evolved as intra- or inter-sexual signals of male phenotypic quality. The hypotheses that song rate and song features reflect androgen levels and body condition, qualities useful in male–male competition, and that they are also influenced by social context, was tested for the first time in the present correlational study. The relationships between song rate and 14 variables describing song structure, respectively, and absolute plasma testosterone levels, body mass, body condition, number of neighbouring males and distances between nest sites in male barn swallows,Hirundo rustica, were analysed. Song rate was not correlated with any of the song features nor with male or social context characteristics. By contrast, a harsh song syllable, the ‘rattle’, was positively related to plasma testosterone levels, and its peak amplitude frequency varied inversely with male body mass and condition. In addition, eight features of song varied according to the social environment of each male. In particular, males sang longer and more varied songs when they had few or no neighbours, whereas males in highly competitive contexts uttered short songs, interrupted them more frequently, and emphasized the rattle. Neighbouring males also sang more similar songs than distant males, and this resulted in matched countersinging. The quality of song output therefore reflects aspects of male competitive potential, and relationships between song structure and social context suggest that some features, such as the rattle, might have originally evolved to serve in male–male interactions; a female preference may have further promoted song evolution leading to complex syllable repertoires.     

Forebrain circuits underlying the social modulation of vocal communication signals

Across vertebrate species, signalers alter the structure of their communication signals based on the social context. For example, male Bengalese finches produce faster and more stereotyped songs when directing song to females (female‐directed [FD] song) than when singing in isolation (undirected [UD] song), and such changes have been found to increase the attractiveness of a male's song. Despite the importance of such social influences, little is known about the mechanisms underlying the social modulation of communication signals. To this end, we analyzed differences in immediate early gene (EGR‐1) expression when Bengalese finches produced FD or UD song. Relative to silent birds, EGR‐1 expression was elevated in birds producing either FD or UD song throughout vocal control circuitry, including the interface nucleus of the nidopallium (NIf), HVC, the robust nucleus of the arcopallium (RA), Area X, and the lateral magnocellular nucleus of the anterior nidopallium (LMAN). Moreover, EGR‐1 expression was higher in HVC, RA, Area X, and LMAN in males producing UD song than in males producing FD song, indicating that social context modulated EGR‐1 expression in these areas. However, EGR‐1 expression was not significantly different between males producing FD or UD song in NIf, the primary vocal motor input into HVC, suggesting that context‐dependent changes could arise de novo in HVC. The pattern of context‐dependent differences in EGR‐1 expression in the Bengalese finch was highly similar to that in the zebra finch and suggests that social context affects song structure by modulating activity throughout vocal control nuclei. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 47–63, 2016     

Testosterone-induced Singing in Female European Starlings (Sturnus vulgaris)

Adult female starlings (Sturnus vulgaris) were injected with testosterone in order to determine the song structures they had in memory. Those females had been caught in the wild in different geographical areas and had different ages. This hormone treatment clearly stimulated singing behaviour of females in isolation and revealed their ability to sing quite complex songs. Both categories of songs observed in males (warbling and whistles) were demonstrated and large repertoires were observed. Although the testosterone treatment may have altered the female song system and induced male-like vocalizations, it is interesting that none of the species-typic elements of male starlings was produced by the experimental females. Large changes were found in both repertoire size and composition over a year, revealing a high plasticity, even in older birds. We also found evidence that even under testosterone treatment, females do not necessarily sing all the songs they may have in memory.