Simultaneous treatment with an aromatase inhibitor and an anti-androgen decreases the likelihood of dawn song in free-living male great tits, Parus major

Gonadal steroids, most importantly testosterone (T), are considered to be a major factor in the expression of adult song behavior in temperate-zone songbirds. The action of T within specific brain regions involved in the regulation of song may occur either directly, or through its androgenic or estrogenic metabolites. In the present study, we tested steroid-dependence of great tit dawn song by blocking both known pathways of T action by simultaneous implantation of flutamide, an anti-androgen, and ATD, an aromatase inhibitor. By our knowledge, this is the first study investigating the effects of androgen inhibitors on dawn song in free-living birds. Male great tits were implanted during their mate's egg laying stage, being the time of maximal male song activity at dawn. Treatment with ATD and flutamide significantly increased plasma T levels, probably because feedback mechanisms on T secretion were inhibited. The treatment decreased the likelihood of showing dawn song, which is in line with the hypothesis that sex steroids are involved in the endocrine control of song behavior. In males that did show dawn song, we found no evidence for a treatment effect on song quality. Although the implants were present for the larger part of the breeding season, males were able to maintain control of a territory and mate and to complete their brood cycle as successful as control males.     

Seasonal activation and inactivation of song motor memories in wild canaries is not reflected in neuroanatomical changes of forebrain song areas

Seasonal, testosterone-dependent changes in sexual behaviors are common in male vertebrates. In songbirds such seasonal changes occur in a learned behavior--singing. Domesticated male canaries (Serinus canaria) appear to lose song units (syllables) after the breeding season and learn new ones until the next breeding season. Here we demonstrate in a longitudinal field study of individual, free-living nondomesticated (wild) canaries (S. canaria) a different mode of seasonal behavioral plasticity, seasonal activation, and inactivation of auditory-motor memories. The song repertoire composition of wild canaries changes seasonally: about 25% of the syllables are sung seasonally; the remainder occur year-round, despite seasonal changes in the temporal patterns of song. In the breeding season, males sing an increased number of fast frequency-modulated syllables, which are sexually attractive for females, in correlation with seasonally increased testosterone levels. About 50% of the syllables that were lost after one breeding season reappear in the following breeding season. Furthermore, some identical syllable sequences are reactivated on an annual basis. The seasonal plasticity in vocal behavior occurred despite the gross anatomical and ultrastructural stability of the forebrain song control areas HVc and RA that are involved in syllable motor control.     

Experimentally Elevated Plasma Testosterone Levels Do Not Influence Singing Behaviour of Male Blue Tits (Parus caeruleus) During the Early Breeding Season

Sexual selection theory suggests that females might prefer males on the basis of testosterone (T)‐dependent secondary sexual traits such as song. Correlational studies have linked high plasma T‐levels to high diurnal song output. This has been confirmed in experiments where T‐levels were kept high at times when natural T‐levels have decreased. However, surprisingly little is known about the relation between T‐levels during the early breeding season and song. In many passerine birds males sing at a high rate at dawn early in the breeding season, referred to as the dawn chorus. In blue tits (Parus caeruleus), the dawn chorus coincides with the fertile period of the female, whereas diurnal song occurs throughout the breeding season. Previous studies on blue tits showed that characteristics of the dawn chorus correlate with male reproductive success. We experimentally elevated plasma T‐levels in male blue tits during the pre‐fertile and fertile period. Our aim was to test whether increased plasma T‐levels affect dawn song characteristics and increase the amount of diurnal song. Although T‐implants successfully raised circulating T‐levels, we did not find any difference between T‐ and control males in temporal performance measures of dawn song or in diurnal song output. Our results suggest that either there is no direct causal link between song output or quality and individual T‐levels, or experimental manipulations of T‐levels using implants do not permit detection of such effects during the early breeding season. Although we cannot exclude that individual T‐levels are causally linked to other (e.g. structural) song parameters, our results cast doubt on T‐dependence as the mechanisms that enforces honesty on song as a sexually selected trait.     

Plasma levels of prolactin and gonadal steroids in relation to multiple-brooding and renesting in free-living populations of the song sparrow, Melospiza melodia

Plasma levels of testosterone (T), estradiol (E2), and prolactin were measured in samples collected from free-living song sparrows, Melospiza melodia. In males, plasma levels of T were elevated early in the season when territories were established and when females laid the first clutch of eggs. Thereafter, T levels declined and remained low throughout the remainder of the breeding cycle. However, if the first brood was lost to a predator, or by experimental removal of the nest, plasma levels of T increased as renesting, to replace the clutch, occurred. Circulating levels of prolactin in males began to rise during the egg-laying stage of the first brood, reached a maximum toward the end of the incubation stage, remained elevated until breeding was terminated, and then declined throughout the moulting stage to basal values in October. Prolactin levels remained high throughout the breeding season irrespective of whether a brood was raised successfully or whether the nest was lost and renesting occurred. In females, plasma levels of E2 were elevated prior to the egg-laying stage for each brood as is typical of multiple-brooded species. However, prolactin titers rose dramatically during egg-laying for the first clutch (slightly higher than in males) and were maximal by onset of incubation. Only females of this species incubate, although males do feed young. As in males, plasma prolactin in females remained high between broods and during experimentally induced renesting, and then declined to basal by the end of the moult stage in October. These data suggest that there are no differences in the temporal patterns of prolactin concentrations in blood between multiple-brooding and renesting. In a separate experiment, captive male song sparrows were transferred from a short day to artificial long days (18L 6D) and a control group was maintained on 9L 15D. In the long-day group, prolactin levels rose abruptly over the first 20 days, as the testes developed, and remained high well into postnuptial moult after the gonads had regressed. Prolactin remained basal in the control group. These data suggest that the temporal pattern of circulating prolactin levels throughout the breeding season is regulated at least partly by changing photoperiod. However, nonphotoperiodic factors are also important since photoperiodically induced increases in prolactin are significantly less than those seen in free-living individuals. These differences may be related to parental behavior.     

Hatching late in the season requires flexibility in the timing of song learning

Most songbirds learn their songs from adult tutors, who can be their father or other male conspecifics. However, the variables that control song learning in a natural social context are largely unknown. We investigated whether the time of hatching of male domesticated canaries has an impact on their song development and on the neuroendocrine parameters of the song control system. Average age difference between early- and late-hatched males was 50 days with a maximum of 90 days. Song activity of adult tutor males decreased significantly during the breeding season. While early-hatched males were exposed to tutor songs for on average the first 99 days, late-hatched peers heard adult song only during the first 48 days of life. Remarkably, although hatching late in the season negatively affected body condition, no differences between both groups of males were found in song characteristics either in autumn or in the following spring. Similarly, hatching date had no effect on song nucleus size and circulating testosterone levels. Our data suggest that late-hatched males must have undergone accelerated song development. Furthermore, the limited tutor song exposure did not affect adult song organization and song performance.     

Breeding Experience and not Age Modulates the Song Development of Pied Flycatchers (Ficedula hypoleuca)

In songbirds, the development of the species‐specific adult song involves a learning process that varies in extension. In species that incorporate new song elements throughout life (open‐ended learners), variation in male song composition could be the result of either age or breeding experience. Using data from 16 yr of fieldwork on pied flycatchers (Ficedula hypoleuca), we aimed to disclose the individual contribution of these two factors on the species song characteristics, as well as their relation with morphology and plumage color changes. Finally, we explored whether any of the song or physical features could predict the probability of males returning to the breeding site. We found that the song characteristics of the first‐time breeders did not differ between age classes, except for the total number of syllables per song, which was higher in the 1‐year‐old than in the 2‐year‐old males. However, we found that song variables associated with complexity (song and sample versatilities and repertoire size), increased significantly from the first to the second breeding season. Males showed delayed plumage maturation, with 1‐year‐old males being browner than the 2‐year‐old males independently of their breeding experience. Morphology, however, was not affected by age or breeding experience. The probability that males returned to the breeding site was not associated with song or physical features. Considering that some song learning occurs during the breeding period and that some males may skip the first breeding season, selective pressures may have been established for song complexity to be an honest indicator of breeding experience rather than age.     

Estrogen contributes to seasonal plasticity of the adult avian song control system

Songbirds show dramatic neural plasticity as adults, including large-scale anatomical changes in discrete brain regions ("song control nuclei") controlling the production of singing behavior. The volumes of several song control nuclei are much larger in the breeding season than in the nonbreeding season, and these seasonal neural changes are regulated by plasma testosterone (T) levels. In many cases, the effects of T on the central nervous system are mediated by neural conversion to estradiol (E(2)) by the enzyme aromatase. The forebrain of male songbirds expresses very high levels of aromatase, in some cases adjacent to song control nuclei. We examined the effects of aromatase inhibition and estrogen treatment on song nuclei size using wild male songbirds in both the breeding and nonbreeding seasons. In breeding males, aromatase inhibition caused the volume of a telencephalic song control nucleus (HVC) to decrease, and this effect was partially rescued by concurrent estrogen replacement. In nonbreeding males, estradiol treatment caused HVC to grow to maximal spring size within 2 weeks. Overall, these data suggest that aromatization of T is an important mediator of song control system plasticity, and that estradiol has neurotrophic effects in adult male songbirds. This study demonstrates that estrogen can affect adult neural plasticity on a gross anatomical scale and is the first examination of estrogen effects on the brain of a wild animal.     

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.     

Context-dependent effects of castration and testosterone treatment on song in male European starlings

Most seasonally breeding songbirds display dramatic seasonal fluctuations in plasma testosterone (T) levels and mate attraction behaviors, including song. However, males of some songbird species, such as the European starling (Sturnus vulgaris), continue to sing at high levels after the breeding season, when T levels are basal. In male starlings song during the breeding season functions mainly to attract mates, whereas song during the nonbreeding season appears unrelated to reproduction. This suggests that song produced in a context unrelated to female courtship, unlike song directed toward females, is not regulated by plasma T. In captive males housed in large outdoor aviaries we explored the relationship between plasma T and song produced during the breeding season within and outside a courtship context. This was achieved by determining the effects of castration and subsequent T treatment on song and mate attraction behaviors in both the presence and the absence of a female. Compared to intact males, castrated males did not show reduced song activity in the absence of a female for at least 6 months after the operation, strongly suggesting that the expression of noncourtship song is not regulated by plasma T. Likewise, we found that experimentally elevating T levels in castrated males did not affect noncourtship song rates. However, control castrated males receiving empty implants tended to show reduced noncourtship song rates after implantation. This may have been due to a suppressive effect caused by the presence of the T-implanted castrated males in the same aviary. In contrast, courtship singing was clearly controlled by plasma T: it was abolished by castration and restored by subsequent T replacement when males were housed both individually and in a group situation. High plasma levels of T also appeared necessary for the activation of three other behavioral traits critical for mate attraction, namely, nesthole occupancy, spending time (singing) in a nesthole, and carrying green nesting material into a nesthole.     

Hormonal correlates of parental behavior in yellow-eyed penguins (Megadyptes antipodes)

Penguins show varying degrees of brood reduction behavior, from obligate brood reducers to brood maximizers, and we hypothesize that this is associated with differences in prolactin secretion. To address this hypothesis, we determined the breeding season prolactin profile of the yellow-eyed penguin (Megadyptes antipodes) for comparison with those of other penguin species found in the literature. We also measured sex steroid plasma concentrations to better characterize the reproductive cycle of the species. Plasma concentrations of prolactin increased from early in the season, reaching a peak during late incubation, and remained elevated up to the guard period. This general pattern was similar to that of other penguins for which we have corresponding data. However, we found that throughout the laying period, prolactin titers in yellow-eyed penguins remained elevated while they fell to basal levels after the laying of the first egg in macaroni penguins, which corresponds to differences in incubation behavior during this time. We conclude, therefore, that differences in the brood reduction behavior in penguins, may be reflected in the pattern of PRL concentrations around the time of egg laying.     

Temporal patterns of circulating LH and steroid hormones in a brood parasite, the Brown-headed cowbird, Molothrus afer.

The breeding behaviour of female Brown-headed cowbirds, Molothrus ater , is remarkably distinct from that of females of most other passerine species. Cowbirds are brood parasites that do not build nests, incubate eggs or feed young. Instead, they have an extended egg-laying stage, and deposit eggs in the nests of hosts within large breeding territories that are defended from other female cowbirds. In this communication, we report the changes in plasma levels of circulating hormones and in morphological characteristics that occur in free-living female Brown-headed cowbirds over a breeding season.
Plasma LH in female cowbirds increases at the onset of breeding and remains elevated for approximately eight weeks, at which time levels decline to basal values. This pattern is similar to that of follicle development, and is consistent with the prolific reproductive output by individuals of this species. Circulating E, levels are quite variable and do not demonstrate any discernible pattern, while plasma B is elevated during the egg-laying period and declines subsequently.
Testosterone titre (T) is highest early in the breeding season, when territorial interactions are generally most frequent. T levels decline slowly thereafter, reaching their lowest values at the season's end. Plasma DHT levels are slightly higher than those for T; DHT exhibits little change until late in the breeding season, when it declines. Fat scores are highest prior to egg laying, and fat reserves diminish as the breeding season progresses. In contrast, body weight increases during the reproductive season, probably as a result of developing eggs in the ovary. These results are consistent with the hypothesis that temporal patterns of hormone secretion are correlated with the expression of social systems.     

Hormone profiles of obligate avian brood parasites during the breeding season

The ultimate explanations for avian brood parasitism have been studied intensively as a model system for coevolution, but little is known about the proximate mechanisms, for example hormonal regulation, underlying brood parasitic behaviour. In this study, we explored seasonal hormone profiles in two brood parasitic Cuculus species breeding in the Republic of Korea. As brood parasites have relatively simple breeding stages without incubation and provisioning, we predicted that during the breeding season individuals would exhibit similar levels of testosterone (T) and stress‐induced corticosterone (CORT), hormones that are known to be closely related to the transition of breeding stages. We also assessed how these hormone profiles were associated with traits such as body size and sex. Overall, male cuckoos showed similarly high T levels throughout the breeding season, as predicted, but individual variation became greater as the season progressed. Individual CORT levels tended to decrease as the season progressed, although the decrease was significant only in Common Cuckoos Cuculus canorus. We also found that male Lesser Cuckoos Cuculus poliocephalus showed a much higher level of T than females, as expected, but this sexual difference was not observed in Common Cuckoos. Our results suggest that the seasonal hormone profiles of avian brood parasites are likely to be similar to typical hormone profiles expected for non‐brood parasites during the breeding season. This may suggest that not only the breeding cycle but also other factors such as social interaction may be affected by hormonal changes. Further studies are needed to fully understand the proximate mechanism of avian brood parasitism.     

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.     

Temporal patterns of circulating LH and steroid hormones in a brood parasite, the Brown-headed cowbird, Molothrus ater.

The reproductive cycle of monogamous passerines is characterized by a sequence of events leading from courtship and pair formation through nest building, egg laying and incubation, and ending with the care of young. Each of these stages is accompanied by equally predictable changes in the pattern of hormone secretion. This report describes endocrine changes in the plasma of free-living Brown-headed cowbirds, Molothrus uter , during a breeding season. Changes in some morphological parameters are also presented.
Cowbirds are brood parasites that show no parental behaviour and lay eggs almost daily for approximately eight weeks. Males are not territorial, but they mate guard extensively. Hcncc, their breeding biology is quite different from that of the nesting species described above. They are hormonally distinct, as well. Luteinizing hormone rises in the spring and remains elevated throughout May and most of June. By July, when breeding has ceased, plasma luteinizing hormone levels are at pre-breeding values. Plasma androgens are also maintained at peak levels for an extended period of time, although they decline briefly between the establishment of dominance hierarchies and the onset of breeding. Circulating levels of corticosterone are elevated until the middle of the breeding season, when they begin to fall, although here, too, there is a transient depression just prior to breeding. In the light of the studies on nesting species, these data suggest that hormone profiles are closely correlated with the breeding and social activities that characterize the species under investigation.     

Lesions targeted to the anterior forebrain disrupt vocal variability associated with testosterone‐induced sensorimotor song development in adult female canaries,Serinus canaria

Learned communication was a trait observed in a limited number of vertebrates such as humans but also songbirds (i.e., species in the suborder passeri sometimes called oscines). Robust male‐biased sex‐differences in song development and production have been observed in several songbird species. However, in some of these species treating adult females with testosterone (T) induced neuro‐behavioral changes such that females become more male‐like in brain and behavior. T‐treatment in these adult females seemed to stimulate sensorimotor song development to facilitate song masculinization. In male songbirds it was known that the lateral magnocellular nucleus of the anterior nidopallium (LMAN) played a modulatory role during song development. LMAN was androgen sensitive and may be a key target of a T‐induced recapitulation of a developmental process in adult females. This hypothesis was tested. Adult female canaries were given either a chemical lesion of LMAN or a control sham‐surgery. Prior to surgery birds were individually housed for 2‐weeks in sound‐attenuated chambers to record baseline vocal behavior. Post‐surgery birds were given 1‐week to recover before subcutaneous implantation with silastic capsules filled with crystalline‐T. Birds remained on treatment for 3‐weeks (behavioral recordings continued throughout). Birds with a lesion to LMAN had less variability in their song compared with controls. The diversity of syllable and phrase type(s) was greater in sham controls as compared with birds with LMAN lesions. Birds did not differ in song rate. These data suggested that the sustention and conclusion of T‐induced sensorimotor song development in adult female canaries required an intact LMAN. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 3–18, 2016     

The Return of the Intruder: Immediate and Later Effects of Different Approach Distances in a Territorial Songbird

Male songbirds often maintain territories throughout the breeding season, and one of the main functions of song is to deter invaders. Therefore, the distance of an unknown singing rival should play a crucial role within territorial singing interactions of males. This distance is expected to be assessed as more threatening the closer the rival approaches. Here, we tested this assumption by conducting nocturnal playbacks from two different distances in territorial Common nightingales (Luscinia megarhynchos). Immediate vocal responses of birds were examined by analysing changes in song structure as well as temporal response features. The next morning, follow‐up playbacks from an intermediate distance allowed us to investigate longer‐lasting effects of nocturnal playbacks. We found that the distance of a simulated rival had an effect on both immediate and later vocal responses of territorial male nightingales with different song parameters being affected during nocturnal and diurnal singing. This indicates that birds perceive intruder distances and adjust their response behaviour both immediately and in later interactions.     

Testosterone, cuckoldry risk and extra-pair opportunities in the Seychelles warbler

In male birds, testosterone (T) plays an important role in aggressive and mate-attraction behaviour. In the cooperatively breeding Seychelles warbler, Acrocephalus sechellensis, extra-group copulations (EGCs) occur frequently, but are not accompanied by sexual courtship displays as in within-pair copulations. Paternity is nearly always gained by primary males. We investigated whether T levels and sperm storage capability (cloacal protuberance (CP)) in adult primary and subordinate males were related to timing of egg laying, levels of cuckoldry and extra-group paternity (EGP) opportunities. During the sexually active period before egg laying, T levels and CP were only elevated or enlarged (respectively) in primary males, and some suggestion was found that subordinate males do not invest in elevated T levels. The peak in T occurred during the fertile period of the female partner and corresponded to the peak period of male sexual displays and mate guarding, but was independent of cuckoldry risk (density of neighbouring primary males). CP was also enhanced during this period; however, CP but not T remained elevated after egg laying by their mates, and CP but not T was positively related to EGP opportunities (density of neighbouring fertile females). We conclude that T is involved in sexual courtship displays and mate guarding, but not in gaining EGCs. These findings contrast with those in other species where EGP involves elaborate sexual displays.     

Temporal Variation in Decisions about Parental Care in Bluegill, Lepomis macrochirus

Parental investment theory states that an individual will trade‐off present and future reproductive potential to maximize lifetime reproductive success. Only when parental care is costly in terms of reduced future reproductive potential should individuals be sensitive to changes in the value of current offspring and adjust their care. Here, we examine temporal variation in parental care decision‐making in bluegill (Lepomis macrochirus), in which care is provided by males called ‘parentals’. Previous research has shown that parentals that nest early in the breeding season are in higher energetic condition than those that nest later, and early nesting males appear not to pay an opportunity cost to their care in terms of reduced future reproductive potential. Early nesting males also may have higher paternity in their broods than later nesting males. To examine the parental care decisions made by early and mid‐season nesting parentals, we experimentally reduced males’ perceived paternity by swapping eggs between nests. We found that experimental males that nested early in the breeding season adjusted their brood defence behaviour similarly to control males, which had sham egg swaps performed. Conversely, experimental males that nested mid‐season significantly decreased their brood defence behaviour after the manipulation as compared with control males. Thus, unlike mid‐season nesting males, early nesting males appear relatively insensitive to changes in brood value (paternity), possibly because early nesting males pay little cost in terms of reduced future reproductive potential to providing full care or because these males have a predisposition to high paternity.     

Seasonality in song behaviour revisited: seasonal and annual variants and invariants in the song of the domesticated canary (Serinus canaria)

The song of the domesticated canary (Serinus canaria) is one of the most widely used models to study the neural correlates of behavioural plasticity and the mechanisms of female mate choice. However, only few studies have described the song behaviour in detail and monitored their changes throughout the year, and these data are restricted to the “Waterslager” strain. Here, we studied the song characteristics of the male common domesticated canary at different times of the year, the spring breeding and autumnal non-breeding season, and monitored the birds' songs up to the following breeding season. During breeding, males have increased plasma levels of testosterone, and songs are on average longer and consist of fewer non-repeated syllable types compared to the non-breeding season. When subsequent seasons are compared, song duration and the proportion of non-repeated syllable types change seasonally but not across years. Repertoire size remains constant throughout seasons although syllable types are exchanged. Syllable carry-over is significantly higher from one breeding season to the next than between the breeding and non-breeding season. Further, the repertoire of the breeding season contains more potentially sexually attractive syllable types than that of the non-breeding season. These data show that overall song structure is retained throughout the year while seasonality occurs in the temporal pattern and in repertoire composition.     

Endocrine correlates of parental care in an Antarctic winter breeding seabird, the emperor penguin, Aptenodytes forsteri

Plasma levels of luteinizing hormone (LH) and prolactin associated with parental behavior were measured in the Antarctic winter breeding emperor penguin, Aptenodytes forsteri. Males exclusively incubate the egg while females exclusively brood the nonhomeothermic young; both sexes alternate in rearing the homeothermic young. Birds were sampled on arrival from the sea through egg laying, incubation, and brooding. All parent birds lost their chicks at the end of the brooding period due to harsh weather but sampling continued. In females, LH titers dropped after egg laying but levels were restored when the birds returned from the sea to brood the chicks and were not depressed by high prolactin levels. Plasma prolactin remained low in males captured on arrival and kept until the free-living males finished incubation. In breeders, prolactin secretion increased during the prelaying period when day length decreased. Prolactin levels stayed elevated in males during incubation and in brooding females returning after a 2-month absence at sea. Prolactin values were higher in brooding females than in males ending incubation or returning in late brooding. These levels did not drop after chick loss, and the sexual difference in prolactin values was maintained after breeding failure. In emperor penguins, increased prolactin secretion appears to be triggered around the time of egg laying and continues, driven by an endogenous mechanism, through incubation and brooding until rearing is completed. Prolactin secretion independent of external stimuli may have evolved in pelagic seabirds to maintain parental care despite long absences at sea from the breeding colony.