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.

Regulation of yolk-androgen concentrations by plasma prolactin in the American kestrel.

The concentrations of maternally derived androgens in the yolks of avian eggs vary within and among clutches, but a mechanistic basis for this variation has not been elucidated. We investigated in the American kestrel, Falco sparverius, whether changes in plasma-prolactin concentrations induced by changes in photoperiod and food supply affect yolk-androgen concentrations. Over the course of a photoinduced breeding period in the laboratory, we measured concentrations of plasma immunoreactive prolactin (ir-prolactin) in female kestrels with ad libitum food availability (control) or food availability that was reduced during the early breeding period. In a second laboratory study, we administered via osmotic mini-pumps ovine prolactin (o-prolactin) to females beginning on the day they laid their first egg of a clutch (egg-day 1) to determine the effects of high prolactin concentrations on yolk-androgen concentrations. In both this study and one on free-living kestrels, we quantified changes in yolk-androgen concentrations with date of clutch initiation. Concentrations of ir-prolactin in nonlaying females rose with date, irrespective of food treatment. Egg-day 1 ir-prolactin concentrations were higher in control females laying late during the breeding phase than in those laying early. This increase was absent in food-reduced females. Yolk-androgen concentrations in eggs 3 and 4 but not eggs 1 and 2 of the clutch were higher in clutches initiated late than in clutches initiated early in the breeding phase in both the field and laboratory. o-prolactin treatment elevated yolk-testosterone but not androstenedione concentrations. These findings suggest that, in American kestrels, seasonal and laying-associated increases in plasma-prolactin concentrations elevate yolk-testosterone concentrations. Food availability and other factors may interact with date to regulate the effects of prolactin on yolk-testosterone deposition.

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.

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.

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.

Maternal steroids and contaminants in common tern eggs: a mechanism of endocrine disruption?

We looked for evidence for the hypothesis that exposure of female birds to polychlorinated biphenyls (PCBs) results in alteration of blood steroid hormone concentrations and alters subsequent hormone transfer of steroids to eggs. Eggs of three-egg clutches were collected from a PCB-exposed common tern (Sterna hirundo) colony (Ram Island, Buzzards Bay, MA, USA) and from a relatively clean colony (Bodkin Island, Chesapeake Bay, MD, USA), and were analyzed for concentrations of organochlorine contaminants and steroid hormones (17beta-estradiol, 5alpha-dihydrotestosterone, testosterone and androstenedione). There was no relationship between total PCBs and steroid concentrations considering all eggs together, considering eggs of different laying order or considering differences between sequentially laid eggs in a clutch. Similarly, concentrations of di- and tri-chlorinated biphenyls and steroids in eggs were not related. The concentrations of PCBs, mercury and selenium were below estimated thresholds for toxicity to embryos. Maternal steroids, except estradiol, were present in yolk of all eggs, with increasing concentrations in the second and third eggs laid. Our data provided no evidence for a maternal toxicological event that might alter the amount of maternal steroid hormone transferred to eggs.

The role of prolactin in the regulation of clutch size and onset of incubation behavior in the American kestrel.

In most bird species, the timing of incubation onset may influence the degree of hatching asynchrony, which, together with variation in clutch size, affects reproductive success. In some domesticated species that usually show no hatching asynchrony, plasma prolactin concentrations in females rise with the onset of incubation and the end of laying, and this rise enhances incubation behavior and may terminate laying. To investigate whether a rise in prolactin during laying is involved in the regulation of clutch size and incubation onset in a species with hatching asynchrony, we measured plasma concentrations of immunoreactive prolactin (ir-prolactin) in laying American kestrels, Falco sparverius, and quantified clutch size and incubation behavior. In a separate study, we administered one of three concentrations of ovine prolactin (o-prolactin) via osmotic pumps implanted in females when egg 2 of a clutch was laid. ir-Prolactin concentrations during laying were higher in small than in large clutches and increased in parallel with the development of incubation behavior. o-Prolactin treatment enhanced incubation behavior, but did not affect clutch size, possibly because the manipulation was performed after clutch size had already been determined. Consistent with studies on domesticated species that show synchronous hatching, our results indicate that rising prolactin during laying enhances the expression of incubation behavior in a species that shows hatching asynchrony. Further studies are necessary to determine whether the relationship between prolactin and clutch size in the American kestrel is one of causation or of mere association.

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.

Regulation of protein breakdown and adrenocortical response to stress in birds during migratory flight.

During long-term fasting at rest, protein utilization is maintained at low levels until it increases at a threshold adiposity. This study examines 1) whether such a shift in energy substrate use also occurs during endurance exercise while fasting, 2) the role of corticosterone, and 3) the adrenocortical response to an acute stressor. Ten species of migrating birds caught after an endurance flight over at least 500 km were examined. Plasma uric acid and corticosterone levels were low in birds with fat stores >5% of body mass and high in birds with smaller fat stores. Corticosterone levels were very high in birds with no visible fat stores and emaciated breast muscles. Corticosterone levels increased with handling time only in birds with large fat stores. These findings suggest that 1) migrating birds with appreciable fat stores are not stressed by endurance flight, 2) a metabolic shift (increased protein breakdown), regulated by an endocrine shift (medium corticosterone levels), occurs at a threshold adiposity, as observed in birds at rest, 3) adrenocortical response to an acute stressor is inhibited after this shift, and 4) an adrenocortical response typical for an emergency situation (high corticosterone levels) is only reached when muscle protein is dangerously low.

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.

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.

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.

Context determines the sex appeal of male zebra finch song.

We explored the conditions under which playbacks of male zebra finch, Taeniopygia guttata, song induced reproduction in females. In a laboratory study, a rise in faecal oestrogen levels predicted egg laying. Song playbacks by themselves induced a decrease in oestrogen levels. There was an increase in oestrogen levels, followed by egg laying, when the song was broadcast from inside a male model positioned away from the nest. However, this effect occurred only when a second, silent male model was perched on the rim of the nest. If song was broadcast from inside the model perched on the nest, there was no increase in oestrogen levels. We conclude that tests of song efficacy in female songbirds must respect some contextual rules, which are likely to vary between species. Only then does it become possible to ascertain which sounds are most effective in inducing physiological changes leading to reproduction. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

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.

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.

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.

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.