Effects of prenatal testosterone on cumulative markers of oxidative damage to organs of young adult zebra finches (Taeniopygia guttata).

We tested the hypothesis that exposure of avian embryos to androgens in ovo entails long-term costs in the form of oxidative damage to vital cells and organs in adulthood. We injected zebra finch eggs with testosterone (T), monitored postnatal growth, and analyzed markers of oxidative damage in heart and liver in mature birds. We measured 8-oxo-2'-deoxyguanosine and isoprostanes, markers of oxidative damage to DNA and membrane lipids, respectively. T treatment (1) reduced growth rates of female but not male nestlings vs. controls; (2) resulted in less accumulation of 8-oxo-dG, but not IsoPs, in liver tissue of 60-day-old females, but not males; and (3) a trend toward elevated 8-oxo-dG levels in heart tissue of males and females at 60 and 180 days old combined. These results generally support the testosterone oxidative damage hypothesis, in that embryonic exposure to higher T resulted in damage to DNA of heart tissue in both sexes. They also suggest that sex-specific effects of androgens on early growth rates may carry over as differences in some forms of oxidative damage in adults. This supports a basic tenet of evolutionary aging theory that developmental influences early in life can be linked to costs later on.

Yolk steroids in great tit Parus major eggs: variation and covariation between hormones and with environmental and parental factors.

ABSTRACT: Avian mothers can potentially alter the phenotypes of their offspring by varying the concentration of steroid hormones in their eggs. We explored variation in androstenedione (A4), testosterone (T), 5α-dihydrotestosterone (DHT), 17ß-estradiol (E2), and corticosterone (CORT) in the yolks of 12 free-living great tit Parus major clutches. We analyzed variation and covariation in greater detail than previous studies, using models for variation with laying sequence that take into account variable clutch size and comparing correlations between pairs of hormones at the within- and between-clutch levels. We also investigated relationships between hormone levels and various environmental, life history, and parental traits. For three of the five steroids, we found no significant correlates, but based on individual statistical tests (a) DHT varied between clutches with male age (1 year old vs older); (b) DHT and CORT were negatively correlated within clutches with the average temperature on the day (DHT and CORT) or 3 days (DHT only) preceding laying; and (c) DHT in the last egg of the clutch relative to the clutch mean was positively correlated with the interval between clutch completion and the onset of incubation (incubation delay). Relationships with ambient temperature and incubation delay have not previously been reported for any yolk hormone in birds. Intriguingly, the three relationships for DHT are consistent with more DHT being transferred to eggs in situations that could be more energetically challenging for the female. More research is needed to determine the generality of the patterns we found and to understand their functional significance. SIGNIFICANCE STATEMENT: The yolks of birds' eggs contain steroid hormones produced by the mother which can affect the development and behavior of the resultant chicks. We analyzed five steroid hormones in the yolks of wild great tits and show for the first time that yolk hormone levels are related to ambient temperature in the day(s) just before laying and, in the last-laid egg, with the day it is laid relative to the onset of incubation, and that the concentrations of pairs of yolk hormones can vary with each other in a different way between and within clutches. These results contribute insights into the ways in which yolk hormones may adaptively modify the chicks or may reflect physiological processes occurring in the mother.

Yolk androgens reduce offspring survival.

Females may favour some offspring over others by differential deposition of yolk hormones. In American kestrels (Falco sparverius), we found that yolks of eggs laid late in the sequence of a clutch had more testosterone (T) and androstenedione (A4) than yolks of first-laid eggs. To investigate the effects of these yolk androgens on nestling 'fitness', we injected both T and A4 into the yolks of first-laid eggs and compared their hatching time, nestling growth and nestling survival with those of first-laid eggs in which we injected vehicle as a control. Compared to controls, injection of T and A4 at a dose intended to increase their levels to those of later-laid eggs delayed hatching and reduced nestling growth and survival rates. Yolk androgen treatment of egg 1 had no effect on survival of siblings hatching from subsequently laid eggs. The adverse actions of yolk androgen treatment in the kestrel are in contrast to the favourable actions of yolk T treatment found previously in canaries (Serinus canaria). Additional studies are necessary in order to determine whether the deposition of yolk androgens is an adaptive form of parental favouritism or an adverse by-product of endocrine processes during egg formation. Despite its adaptive significance, such 'transgenerational' effects of steroid hormones may have helped to evolutionarily shape the hormonal mechanisms regulating reproduction.

Maternal androgens in black-headed gull (Larus ridibundus) eggs: consequences for chick development.

We tested the hypothesis that mother birds counterbalance the negative effects of hatching asynchrony for later-hatched chicks by increasing the yolk androgen concentrations in consecutive eggs of their clutch. In doing so, they may adaptively tune each offspring's competitive ability and, thus, growth and survival. However, evidence in support of this hypothesis is contradictory. The yolk concentrations of maternal androgens in the eggs of black-headed gulls increase significantly with the laying order of the eggs in a clutch. We experimentally tested the functional consequences of this increase on chick development under natural conditions by injecting eggs with either an oil or androgen solution. We created experimental clutches in which androgen levels either stayed constant or increased with laying order while controlling for differences in egg quality by using only first-laid eggs. We then compared development, growth and survival between these broods. Androgen treatment enhanced embryonic development because androgen-treated eggs hatched half a day earlier than controls, while their size at hatching was similar to oil-treated controls. Androgen treatment did not increase chick survival, but it enhanced growth. Androgen-treated, third-hatched chicks had a higher body mass and longer legs than third-hatched chicks that hatched from oil-treated eggs. At the same time, growth of first chicks (which were all oil treated) was reduced by the presence of two androgen-treated siblings, suggesting that yolk androgens enhance the competitive ability of later-hatched chicks. Our results support the hypothesis that transfer of different amounts of androgens to the eggs of a clutch is a mechanism by which mothers maximize their reproductive output.

Variation in hypothalamic gonadotrophin-releasing hormone content, plasma and pituitary LH, and in-vitro testosterone release in a long-distance migratory bird, the garden warbler (Sylvia borin), under constant photoperiods.

Changes in concentrations of hypothalamic gonadotrophin-releasing hormone (GnRH), pituitary and plasma LH, testicular mass, in-vitro release of testosterone, body mass and migratory activity were measured in male garden warblers (Sylvia borin) kept from November to June under a constant photo-period of 12.8 h. Under such conditions garden warblers gradually change from the photorefractory to the photosensitive state and gonadal recrudescence then occurs. Hypothalamic GnRH content was low from December to March, but increased in April to reach the highest levels in June. The spontaneous increase in GnRH was paralleled by increases in pituitary LH content, testicular mass and in-vitro testosterone release. Body mass decreased 1 month and nocturnal activity 2 months before the spontaneous increase in GnRH. Ovine LH increased in-vitro testosterone release over basal release at all times. The results suggest that in garden warblers (1) changes in hypothalamic GnRH content can occur under constant photoperiodic condition, (2) the gradual change from the photorefractory to the photosensitive state is not characterized by a gradual increase in hypothalamic content of GnRH (cf. starlings), and (3) Leydig cells are capable of testosterone release even during the photorefractory state.

Brain gene regulation by territorial singing behavior in freely ranging songbirds.

To investigate the ecological relevance of brain gene regulation associated with singing behavior in songbirds, we challenged freely ranging song sparrows with conspecific song playbacks within their breeding territories. Males responded by approaching the speaker, searching for an intruder and actively singing. In situ hybridization of brain sections revealed significantly higher expression of the transcriptional regulator ZENK in challenged birds than in unstimulated controls in several auditory structures and song control nuclei. We conclude that singing behavior in the context of territorial defense is associated with gene regulation in brain centers that control song perception and production, and that behaviorally regulated gene expression can be used to investigate brain areas involved in the natural behaviors of freely ranging animals.

Effects of food-deprivation on migratory restlessness and diurnal activity in the garden warbler Sylvia borin.

The present study describes the effects of food-deprivation on migratory restlessness and diurnal activity in the garden warbler, a long-distance migrating passerine. Fourteen first-year birds were held from August through March in individual registration cages and exposed to daylength changes experienced by birds that winter at the equator. With the beginning of the fall migratory season the birds increased their body weights due to fat deposition and showed nocturnal migratory restlessness characteristic for this species. After the birds had deposited more than about 4 g of fat they were food-deprived until their body mass had declined to a value about 1 g above fat free mass. Subsequently they were re-fed. This procedure was repeated through fall and winter, beyond the termination of spontaneous fall migratory restlessness. - Food deprivation consistently caused an increase in duration and intensity of nocturnal activity. After the end of spontaneous migratory restlessness and before the onset of winter molt it led to a reinduction of nocturnal activity. When nocturnal activity increased, the duration of diurnal activity decreased, whereas its intensity increased. With regard to the performance of free-living migrants these results suggest (1) that nocturnal migratory activity is enhanced in birds travelling across areas with unprofitable resting places; (2) that nocturnal migratory activity may be reactivated in birds that have settled for the winter in an area with deteriorating food supply; and (3) that the pattern of diurnal activity may change conspicuously when nocturnal migratory activity is enhanced or resumed.

Flavonoids and tannins: plant-based antioxidants with vitamin character.

Persistent activation of the neuroendocrine stress axis is the major cause of a continuous catabolic alteration of the metabolism. This often causes an oxidative stress situation with increased release of O2 and NO radicals and pro-inflammatory cytokines. For the correction of these metabolic states, an adequate supply of plant-based antioxidants, especially flavonoids and tannins, is indicated. These are plant-based polyphenols which, like vitamins cannot be synthesized by the animal organism. Vitamin E in combination with vitamin C and beta-carotene are currently considered worldwide as the standard antioxidative therapy. However, it has recently been shown that, depending on the iron status of the recipient, pharmacological doses of these vitamins sometimes have beneficial, but often also no effect or harmful effects, so that, for a more reliable antoxidative action, adequate dietary supply of a mixture of flavonoids and tannins seems preferable.

Yolk is a source of maternal testosterone for developing birds.

The sex steroid hormones that affect development in birds have been thought to be produced exclusively by the embryo or neonate. I used radioimmunoassay to measure the amounts of androstenedione, 5 alpha-dihydrotestosterone, testosterone, 17 beta-estradiol, and corticosterone in the yolk of freshly laid canary (Serinus canaria) and zebra finch (Poephila guttata) eggs. Testosterone was found in both canary and zebra finch eggs, but its contents were much higher in the former than in the latter. The testosterone content of canary eggs in a same clutch increased with the order of laying, regardless of the genetic sex of the offspring that hatched from these eggs. Yolk testosterone was also present in the eggs of female canaries that were kept without a male, indicating that it is of maternal origin. The social rank of juvenile canaries was positively correlated with the concentration of yolk testosterone in the eggs from which they hatched, suggesting that the development of aggressive behavior of offspring might be subject to modification by maternal testosterone. These findings indicate that female songbirds can bestow upon their eggs a dose of hormone that modifies the behavior of offspring. Variable doses of these hormones might explain some of the individual variation in offspring behavior.

Developmental changes and among-sibling variation of corticosterone levels in an altricial avian species.

The postnatal development of the activity of the brain-pituitary-adrenal axis was investigated in an altricial bird species by measurements of plasma corticosterone levels in nestling and fledgling canaries, Serinus canaria. Corticosterone was detectable (>2.6 ng/ml) in 30% of 5-day-old, 67% of 10-day-old, 72% of 15-day-old, and 88% of 23-day-old birds. When detectable, the corticosterone levels of 5-day-old nestlings were comparable to the baseline levels of adult birds. Levels were higher in 10- and 15- than in 5-day-old nestlings. The levels of 23-day-old fledglings (about 6 to 7 days after fledgling) were significantly higher than those of 15-day-old nestlings. They were intermediate between adult baseline and stress-induced levels. Sex did not influence this general profile, but levels varied with the order of hatching within broods. At the age of 15 and 23 days first hatched chicks had higher corticosterone levels than last hatched chicks, while second hatched chicks had intermediate levels. These differences were not correlated with body mass. The results suggest that (1) the brain-pituitary-adrenal axis of this altricial bird becomes fully functional after hatching and (2) birth order within broods influences corticosterone secretion during subsequent stages of development. It is unlikely that the brain-pituitary-adrenal axis matures at different rates in first and later hatched chicks or that the different levels of first and later hatched chicks were caused by capture and handling stress. Rather, they may result from such maternal effects as hatching asynchrony or differential concentrations of yolk steroids among the eggs in a clutch. Further studies will have to show whether this systematic variation of corticosterone levels among siblings during early life persists into adulthood and how it is related to behavior and fitness.

Environment modifies the testosterone levels of a female bird and its eggs.

Past studies have shown that the yolk of the canary (Serinus canaria) egg contains maternal testosterone, that its concentrations increase in the subsequently formed eggs of a clutch, and that testosterone influences development. The present study investigated 1) if the testosterone levels vary in the female during yolk formation; 2) how such putative variations may be related to the concentrations of maternal testosterone in the yolk; and 3) if environmental factors, such as day length, can modify the testosterone levels in the mother and her eggs. Maternal testosterone levels, measured in the females' feces, increased during yolk formation and egg laying, and decreased during incubation. This pattern was modified by day length. In a photoperiod of 12 h of light and 12 h of darkness (12L 12D), female testosterone levels decreased after the last egg of the clutch was laid while in a photoperiod of 16L 8D, they decreased after the first egg was laid. These different patterns were reflected in the testosterone concentrations in the egg yolk. Further, the eggs of subsequent clutches that were produced under a naturally changing photoperiod differed significantly in their testosterone concentrations. Finally, the doses of testosterone in the yolk of individual eggs were positively correlated with the concentrations of testosterone in the female during the yolk phase of each egg. I conclude that here we have a mechanism which communicates environmental conditions from the mother to the offspring, and that this mechanism serves to optimize reproduction and/or modify offspring traits.

Maternal testosterone in the avian egg enhances postnatal growth.

The eggs of the canary (Serinus canaria) contain variable doses of maternal testosterone. The reported experiments investigated whether testosterone influences nestling growth and how this interacts with differences of the growth of nest mates that are caused by asynchronous hatching. Injections of testosterone into the yolk of unincubated eggs enhanced the growth after hatching compared to nestlings that had hatched simultaneously from control eggs. These differences were established within 22 hr of hatching. Exogenous testosterone promoted growth in both sexes and there was no sexual difference in the growth of control birds. Testoster-one-treated chicks also begged more often for food. Previous studies have shown that the content of maternal testosterone increases in each subsequently laid egg in a clutch. Consistent with the results obtained by testosterone injections nestlings that hatched from eggs with higher concentrations of maternal testosterone grew faster compared to chicks that hatched synchronously from eggs with lower testosterone concentrations. However, more testosterone did not compensate for reduced growth that was caused by later hatching due to asynchronous incubation of clutches. This direct effect of maternal testosterone on growth in combination with a flexible onset of incubation allows to selectively enhance the growth and fitness of individual offspring of a brood.

Hormonal patterns in breeding and nonbreeding kestrels, Falco tinnunculus: field and laboratory studies.

Plasma levels of luteinizing hormone (LH), androgens, and corticosterone (B) were measured in breeding and nonbreeding kestrels, both in the field and in captivity under different food conditions. LH levels in breeding males were elevated from courtship through incubation and androgen concentration peaked during courtship and laying. Seasonal changes in LH and androgens were similar in breeding and nonbreeding males, although androgens declined sharply after laying in breeding males. Egg laying was characterized by marked increases in both female body mass and plasma concentrations of LH and B. In both breeding and nonbreeding females LH and B increased during pair formation and courtship (March-April), although maximum levels were lower in nonbreeders. Two marked differences were found between free-living and captive (paired) birds. First, during winter plasma levels of LH were basal in free-living birds, while significantly elevated in both captive males and females. Second, during courtship androgen levels in breeding males were three-fold higher in the field than in captivity, probably as a result of intermale aggression under natural conditions. Females breeding early, late, or not at all had similar LH concentrations on arrival and during early courtship. Before breeding there were also no differences in LH (males and females) or in androgen levels (males) between pairs fed ad libitum and pairs temporary food rationed. However, captive females with experimentally reduced food intake showed low levels of B until their rations were increased. Nonbreeders showed hormonal changes similar to breeders, except for those changes in females that were associated with laying. These results suggest that at the start of the breeding season both nonbreeding and breeding kestrels have functional reproductive systems. That some breed early, late, or not at all is primarily an effect of food availability and is not due to hormonal modulation of the reproductive cycle.

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.

Territorial aggression and song of male European robins (Erithacus rubecula) in autumn and spring: effects of antiandrogen treatment.

Male robins aggressively defending a feeding territory in winter have low levels of testosterone, while males defending a breeding territory in spring have elevated levels of testosterone. Song is an integrated part of territorial defense during both phases. We investigated whether testosterone is involved in the expression of these behaviors by treating free-living and captive male robins during both phases with the antiandrogen flutamide. Results suggest that, similar to species in which territoriality is restricted to the reproductive phase, aggressive defense of a breeding territory by male robins is facilitated by androgens. Territorial defense during the nonbreeding season, however, does not require androgenic activity. Singing frequency, on the other hand, was not significantly reduced during either phase by flutamide application. Since the quality of male song changes with season we suggest that the incorporation of sexual signals into male song during breeding depends on testosterone, but that the year-round production of territorial "keep out" signals is independent of testosterone.

Plasma corticosterone in nestling american kestrels: effects of age, handling stress, yolk androgens, and body condition.

The effects of age, handling-induced stress, yolk androgens, and body condition on plasma corticosterone levels were investigated in free-living nestling American kestrels, Falco sparverius, a semialtricial falcon species. In an observational study, corticosterone levels varied with age and handling time. Specifically, corticosterone was low until age 15 days and then rose from age 20 through 25 days. Nestlings as young as age 10 days showed a handling-induced rise in corticosterone. Neither sex nor hatching order of the nestling affected corticosterone levels. Concentrations of maternally derived yolk androgens have previously been shown to be lower in first-laid than in later-laid eggs in the clutch. In an experimental study, androgens were injected into the yolk of the first-laid egg to elevate its levels to those of later-laid eggs, a treatment that substantially reduces nestling body condition compared with that of controls. Yolk androgen treatment elevated posthatching corticosterone levels compared with those of controls, and corticosterone levels were negatively correlated with body condition. These findings indicate that even very young, developing birds can show stress-induced increases in corticosterone and that age-related changes in corticosterone secretion may be modified by body condition and maternal effects such as yolk androgen deposition. The short- and long-term consequences of high glucocorticosteroid levels in young, developing vertebrates are largely unknown.

Photoperiod regulation of neuron death in the adult canary.

The avian brain undergoes naturally occurring cell death and neuronal replacement in adulthood. Little is known about how neuron survival in adult birds is regulated. However, previous work suggests that this process is open to environmental control. We now report that a reduction in day length from spring-like to fall-like conditions can dramatically increase cell death in adult male canaries. Many of the dying cells are projection neurons in the motor pathway controlling song learning and production. Circulating levels of gonadal steroids were not correlated with photoperiod-induced changes in the magnitude of cell death. Our results suggest that neuronal death in adult male canaries is regulated by seasonal changes in photoperiod, and that this occurs independent of chronic changes in gonadal steroid hormone levels. Day length may serve as a predictive environmental cue to time cell death in accordance with seasonal reproduction.

Daily estradiol and progesterone levels relative to laying and onset of incubation in canaries.

Female birds may optimize reproduction by modifying clutch size and the timing for the onset of incubation. We measured fecal estradiol-17beta (E) and progesterone (P) in laying canaries to better understand how onset of incubation might regulate clutch size. Both E and P rose sharply to maxima 1 day before the first egg was laid. Thereafter, E steadily declined, but P remained high through 2 days after the first egg was laid, after which both hormones had returned to low levels. Clutch size did not explain variation in E or P output during the laying cycle. When analyzed with respect to onset of incubation, E and, to a lesser extent, P dropped significantly on the day incubation began, irrespective of whether or not females had finished ovulating. We suggest that factors initiating incubation also cause the decline in E production by small follicles, which in turn may inhibit yolk sequestration in large follicles. Further experiments in which onset of incubation is manipulated may reveal the mechanisms by which this behavior regulates clutch size and reproductive output.

Twenty-four hour melatonin profiles in a nocturnally migrating bird during and between migratory seasons.

The garden warbler Sylvia borin is a long-distance migrating bird that travels exclusively at night. During the migratory seasons caged warblers develop intense nocturnal activity which may become even more intense than that during the day. It is demonstrated that in spite of dramatic seasonal changes in the 24-h pattern of locomotor activity measured in caged garden warblers, the corresponding pattern of plasma melatonin changed only very little. As in other species melatonin levels were generally low during the day (below 50 pg/ml) and high at night (350 to 650 pg/ml). A slight reduction in the nocturnal melatonin peak (from 650 pg/ml to between 350 and 400 pg/ml) observed during the autumn and spring migratory seasons, was possibly due to an increased light perception of the birds moving around while being exposed to dim night lights. In general the results show that the 24-h plasma melatonin profiles of these birds are rather robust and that locomotor activity does not depend in a simple and direct way on plasma melatonin levels.

Maternal investment. Sex differences in avian yolk hormone levels.

It has been suggested that female birds put more resources into eggs fathered by attractive males by laying larger eggs or by adding more testosterone, but this inference could be undermined if eggs of different sex are provisioned differently, as these studies did not control for sex differences. Here we compare hormone concentrations in the yolks of male and female eggs and find that these are significantly different. Our results indicate that it is premature to conclude that female birds invest more in eggs sired by a preferred male, and raise the possibility that yolk sex steroids may be part of the sex-determining process in birds.