Interfemale variation in egg yolk androgen allocation in the European starling: do high-quality females invest more?

Androgenic hormones occur naturally in bird egg yolk and are known to enhance growth in canary and gull chicks. Gil et al. (1999, Science, 286, 126-128) have recently proposed that female allocation of androgens to eggs represents a form of costly maternal investment. This hypothesis predicts that females of high quality or high reproductive potential should invest more yolk androgens in their eggs than females of low quality or low reproductive potential. We tested these predictions by examining interfemale variation in allocation of the androgenic steroids testosterone and androstenedione to eggs in a wild population of European starlings, Sturnus vulgaris. We collected 30 full clutches of eggs and captured 22 of the females that laid these clutches for phenotypic measurements. In agreement with the hypothesis, we found that there was significant interfemale variation in yolk androgen concentrations. Furthermore, older females deposited more androstenedione and testosterone in eggs than 1-year-old females, and females laying early or large clutches deposited more testosterone in eggs than females laying late or small clutches. However, females in good body condition did not deposit more androgens in eggs than females in poor body condition. Large-yolked eggs had a higher total yolk androgen content than small-yolked eggs, but larger eggs did not have higher concentrations of yolk androgens. The results in general support the investment hypothesis of yolk androgen allocation. However, further data are needed regarding the costs of yolk androgen allocation and the effects of yolk androgens on offspring to substantiate the assumptions of the hypothesis. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Maternal androgens in the pied flycatcher: timing of breeding and within-female consistency

Maternal hormones can have substantial phenotypic effects in the progeny of many vertebrates. It has been proposed that mothers adaptively adjust hormone levels experienced by particular young to optimize their reproductive output. In birds, systematic variation in egg hormone levels has been related to different female reproductive strategies. Because in many bird species prospects of the offspring change seasonally and with brood number, strategic adjustment of yolk androgen levels would be expected. To test this idea, we induced pied flycatcher (Ficedula hypoleuca) females to nest twice during the same season by removing their first clutches shortly after clutch completion. We collected eggs of first and replacement clutches to measure yolk concentrations of androstenedione (A4) and testosterone (T) and captured the females that laid these clutches for phenotypic measurements. Although average egg androgen levels were remarkably consistent within females, hormone patterns differed considerably between first and replacement clutches. Eggs of replacement clutches were heavier with larger yolks compared to first clutches, but they contained on average lower levels of androgens. Within clutches, androgen concentration increased over the laying sequence in the first clutch, but decreased or remained more constant over the laying sequence in the replacement clutch. Mean yolk T, but not A4 levels, were negatively associated with laying date for both breeding attempts. Moreover, females in good body condition produced eggs containing lower levels of androgens than females in poor condition. Our results are consistent with the idea that differences in yolk androgen levels may be one mechanism underlying seasonal variation in reproductive success and it is possible that changes in egg androgen patterns may reflect a change in female reproductive strategy. High within-female consistency also highlights the possibility that there may be some underlying genetic variation in yolk androgen levels.     

Pre-breeding diet affects the allocation of yolk hormones in zebra finches Taeniopygia guttata

The ability of mothers to modify offspring phenotype to match prevailing environmental conditions is an important component of reproductive success, especially in variable environments. Pre-breeding conditions, such as food abundance, may have significant consequences for both the number and quality of offspring a female produces as well as her ability to rear the offspring. In an experiment where pre-breeding diet was manipulated, we investigated if allocation of yolk androgens (testosterone and 5α-dihydrotestosterone) was related to the quality of diet experienced prior to breeding. Female zebra finches Taeniopygia guttata produced larger clutches on high quality diet than on low quality diet but with no differences in egg mass. Yolk androgen levels were repeatable within subsequent clutches of the same female and females did not change mean androgen content in eggs in relation to diet quality. However, within-in clutch pattern of yolk testosterone and DHT changed with diet treatment. Testosterone and DHT decreased with laying order on the low quality diet but remained constant on the high quality diet. Differential yolk androgen allocation within the clutch may alter the competitive differences between chicks and provide females the possibility to adjust reproductive investment and offspring phenotype already at the egg stage.     

Maternal androgens in eggs of communally breeding guira cuckoos (Guira guira)

Variation of maternal androgens in avian eggs may be a mechanism of maternal influence on offspring development, growth, and/or behavior. We studied yolk androgen concentrations in eggs of guira cuckoos (Guira guira) to understand how females might enhance the success of offspring in a complex communal breeding system. We measured concentrations of androstenedione, 5alpha-dihydrotestosterone, and testosterone in yolks and identified eggs and clutches of individuals in joint nests by yolk protein electrophoresis. Androstenedione had the highest yolk concentration, at least 10 times higher than that of testosterone and 5alpha-dihydrotestosterone. The first eggs of individual females that laid two or three eggs in a joint nest had lower androstenedione concentrations than their second and third eggs, the latter having a lower probability of being ejected from the nest. This implies that guira cuckoo females may influence offspring survival and competitiveness in communal nests by means of differential allocation of androstenedione and laying tactics. There was significant variation in yolk androstenedione among females, but the order in which females entered laying in the communal clutch had no effect on the concentrations. Androstenedione yolk concentrations increased with communal clutch size, which may indicate that higher levels of competition in larger groups lead to higher yolk androgen concentrations. Finally, androstenedione concentrations were higher in clutches in the later wetter periods of the rainy season than during the earlier drier period. This may be explained by the high frequency of large clutches in the later periods, with more females contributing to a joint clutch.     

Food availability affects the maternal transfer of androgens and antibodies into eggs of a colonial seabird

Mothers can improve the quality of their offspring by increasing the level of certain components in their eggs. To examine whether or not mothers increase deposition of such components in eggs as a function of food availability, we food-supplemented black-legged kittiwake females (Rissa tridactyla) before and during egg laying and compared deposition of androgens and antibodies into eggs of first and experimentally induced replacement clutches. Food-supplemented females transferred lower amounts of androgens and antibodies into eggs of induced replacement clutches than did non-food-supplemented mothers, whereas first clutches presented no differences between treatments. Our results suggest that when females are in lower condition, they transfer more androgens and antibodies into eggs to facilitate chick development despite potential long-term costs for juveniles. Females in prime condition may avoid these potential long-term costs because they can provide their chicks with more and higher quality resources.     

Brood Reduction via Intra-clutch Variation in Testosterone - An Experimental Test in the Great Tit

In birds, yolk androgen concentrations in eggs can increase or decrease over the laying sequence and common hypotheses hold that this serves to favour the competitive ability of either first- or last-hatched chicks depending on the prevailing conditions, and thus promote brood reduction or maintenance of original brood size respectively. Intra-clutch variation of testosterone can shift relative competitive ability of siblings and hence competitive dynamics. In a natural population of great tits, we experimentally investigated the effects and function of maternal testosterone on offspring phenotype in relation to the laying position of the egg in a context of hatching asynchrony. To this end, we created three types of clutches where either the first three or the last three eggs of a clutch were injected with testosterone (T) dissolved in sesame oil, and the remaining eggs with sesame oil only, or where all eggs of a clutch were injected with sesame oil. Increased levels of yolk T in the last-laid eggs resulted in the last-hatched chicks being significantly lighter and smaller than their siblings, while increased levels of T in the first-laid eggs had no direct effect on the first-hatched chicks, but an indirect negative effect on their siblings. Our results suggest that females can potentially adjust offspring phenotype by modulating, over the laying sequence, the amounts of T deposited in the eggs. These results are in contradiction, however, with current hypotheses and previous findings, which suggest that under good conditions higher levels of maternally derived T in the last-laid eggs should mitigate the negative effects of hatching asynchrony.     

Yolk androgens and begging behaviour in black-headed gull chicks: an experimental field study

Black-headed gulls, Larus ridibundus, produce clutches of three eggs, which contain high levels of maternal androgens in the yolk. These levels increase with laying order and the eggs hatch asynchronously. Experiments have supported the hypothesis that this within-clutch variation in maternal androgens mitigates the disadvantage of last-hatched chicks in sibling competition, by enhancing embryonic development and early posthatching growth. We hypothesized that these effects come about by the stimulating effects of maternal androgens on begging behaviour and competitive ability. In the field, we injected first-laid eggs of a clutch (which have a low androgen level) with either an androgen solution (T eggs) or vehicle (Oil eggs). We then created pairs of chicks hatched from Oil and T eggs, matched for egg mass and hatching date. Parent-chick interactions were recorded from observation hides. Chicks from T eggs hatched almost half a day sooner than those from Oil eggs. Furthermore, chicks from T eggs were more active during the first week after hatching, were more often the first to react to the approaching parent, begged more frequently, and obtained the larger share of food. We conclude that the enhancing effect of yolk androgens on growth in this species arises at least partly through androgen-mediated effects on the chicks' behaviour. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.      

The multivariate egg: quantifying within- and among-clutch correlations between maternally derived yolk immunoglobulins and yolk androgens using multivariate mixed models

Egg components are important mediators of prenatal maternal effects in birds and other oviparous species. Because different egg components can have opposite effects on offspring phenotype, selection is expected to favour their mutual adjustment, resulting in a significant covariation between egg components within and/or among clutches. Here we tested for such correlations between maternally derived yolk immunoglobulins and yolk androgens in great tit (Parus major) eggs using a multivariate mixed-model approach. We found no association between yolk immunoglobulins and yolk androgens within clutches, indicating that within clutches the two egg components are deposited independently. Across clutches, however, there was a significant negative relationship between yolk immunoglobulins and yolk androgens, suggesting that selection has co-adjusted their deposition. Furthermore, an experimental manipulation of ectoparasite load affected patterns of covariance among egg components. Yolk immunoglobulins are known to play an important role in nestling immune defence shortly after hatching, whereas yolk androgens, although having growth-enhancing effects under many environmental conditions, can be immunosuppressive. We therefore speculate that variation in the risk of parasitism may play an important role in shaping optimal egg composition and may lead to the observed pattern of yolk immunoglobulin and yolk androgen deposition across clutches. More generally, our case study exemplifies how multivariate mixed-model methodology presents a flexible tool to not only quantify, but also test patterns of (co)variation across different organisational levels and environments, allowing for powerful hypothesis testing in ecophysiology.     

Elevated yolk androgen levels benefit offspring development in a between-clutch context

The field of androgen deposition in avian eggs and its consequencesfor offspring development has received a lot of attention inrecent research. However, although variation within clutchesin yolk androgens is relatively well understood, the adaptivesignificance of patterns of variation between clutches remainsrather unclear. Furthermore, it has been hypothesized that yolkandrogens act as a means of an adaptive maternal effect to adjustoffspring to a given posthatching environment. Thus, the consequencesof maternal yolk androgens for offspring development are likelyto depend on the specific environment of a given brood. We experimentallymanipulated yolk androgen concentrations in spotless starlingeggs, using a between-brood design in which full broods weremanipulated applying either an androgen or sham treatment, inorder to test the effects of between-brood variation in yolkandrogen levels. We also included in the analysis several femalecharacters that have been shown to affect androgen deposition.Androgen-chicks tended to gain more mass, a similar effect tothat shown in previous studies where direct competition betweenchicks belonging to different treatments was allowed, but didnot gain a survival benefit. Androgen-chicks had wider beakflanges, an effect that has not been described previously andcould play an important role in food acquisition. In addition,androgen-chicks had higher endogenous plasma levels of androgens,which could induce higher begging intensity. We hypothesizethat these effects are an important mechanistic link for ourunderstanding of how yolk androgens exert their effects on offspringdevelopment after hatching. Contrary to our predictions, wefound no evidence that the observed effects depended on theenvironment under which it was investigated. Because high yolkandrogen levels seem to be beneficial for nestlings in thisspecies, we hypothesize that yolk hormone deposition could becostly for females or alternatively that potential negativeeffects later in adulthood may constrain maternal hormone allocation.     

Sex-specific effects of yolk testosterone on survival, begging and growth of zebra finches

Yolk androgens affect offspring hatching, begging, growth and survival in many bird species. If these effects are sex-specific, yolk androgen deposition may constitute a mechanism for differential investment in male and female offspring. We tested this hypothesis in zebra finches. In this species, females increase yolk-testosterone levels and produce male-biased sex ratios when paired to more attractive males. We therefore predicted that especially sons benefit from elevated yolk androgens. Eggs were injected with testosterone or sesame oil (controls) after 2 days of incubation. Testosterone had no clear effect on sex-specific embryonic mortality and changed the pattern of early nestling mortality independent of offspring sex. Testosterone-treated eggs took longer to hatch than control eggs. Control males begged significantly longer than females during the first days after hatching and grew significantly faster. These sex differences were reduced in offspring from testosterone-treated eggs due to prolonged begging durations of daughters, enhanced growth of daughters and reduced growth of sons. The results show that variation in maternal testosterone can play an important role in avian sex allocation due to its sex-specific effects on offspring begging and growth.     

Prenatal androgen exposure modulates cellular and humoral immune function of black-headed gull chicks

Avian eggs contain considerable amounts of maternal yolk androgens, which have been shown to beneficially influence the physiology and behaviour of the chick. As androgens may suppress immune functions, they may also entail costs for the chick. This is particularly relevant for colonial species, such as the black-headed gull (Larus ridibundus), in which the aggregation of large numbers of birds during the breeding season enhances the risk of infectious diseases for the hatching chick.To test the effect of maternal yolk androgens on the chick's immune function, we experimentally manipulated, in a field study, yolk androgen levels within the physiological range by in ovo injection of either androgens (testosterone and androstenedione) or sesame oil (control) into freshly laid eggs. We determined cell-mediated immunity (CMI) and humoral immunity of the chicks at the beginning of the nestling period to evaluate early modulatory effects of yolk androgens on immune function.Embryonic exposure to elevated levels of androgens negatively affected both CMI and humoral immunity in nestling gull chicks. Consequently, maternal yolk androgens not only entail benefits of enhanced competitiveness and growth as previously shown, but also costs in terms of immunosuppression. The outcome of embryonic yolk androgen exposure thus likely depends on the post-hatching circumstances for the developing offspring such as parasite exposure and degree of sibling competition.     

Maternal transfer of androgens in eggs is affected by food supplementation but not by predation risk

Mothers may affect the future success of their offspring by varying allocation to eggs and embryos. Allocation may be adaptive based on the environmental conditions perceived during early breeding. We investigated the effects of food supplementation and predation risk on yolk hormone transfer in the pied flycatcher Ficedula hypoleuca. In a food supplementation experiment, females were food‐supplemented prior to and during egg‐laying and androgen concentrations were measured throughout the laying order. Predation risk was investigated in three different studies combining both correlative data, where flycatchers bred in close proximity to two different predator species that prey upon adult flycatchers (either Tengmalm's owl Aegolius funereus or pygmy owl Glaucidium passerinum), and an experimental manipulation, where flycatchers were exposed to cues of a nest predator (least weasel Mustela nivalis). Females receiving food supplementation laid eggs with lower concentrations of androstenedione (A4) than females not receiving food supplements. Yolk testosterone (T) concentration showed the same pattern but the difference was not statically significant. Testosterone (but not A4) concentration increased within clutches, from the first to the last egg, independently of the food supplementation. Females breeding under high predation risk did not differ from control females in their yolk androgen levels (A4, T or progesterone). However, concentrations of A4 tended to be lower in the proximity of pygmy owls, which could indirectly increase offspring survival after fledging. Food supplementation during egg‐laying seems to have a stronger impact on maternal transfer of androgens than predation risk. Food availability and predation risk could differentially affect the trade‐offs of androgen allocation for the offspring when raised in good vs. dangerous environments.     

Maternal dietary carotenoids mitigate detrimental effects of maternal GnRH on offspring immune function in Japanese quail Coturnix japonica

Maternal resources deposited in eggs can affect the development of several offspring phenotypic traits and result in trade‐offs among them. For example, maternal androgens in eggs may be beneficial to offspring growth and competitive ability, but detrimental to immunocompetence and oxidative stress. In contrast, maternal antioxidants in eggs may be beneficial if they mitigate oxidative stress and immunosuppressive effects of androgens. We investigated possible interactive effects of maternal steroids and carotenoids on aspects of offspring physiology and phenotype, by simultaneously manipulating levels of androgens (via gonadotropin‐releasing hormone, GnRH‐challenges) and carotenoids (via diet supplementation) in captive female Japanese quail Coturnix japonica during egg laying. Carotenoid supplementation of hens, which elevates yolk concentrations of carotenoid and vitamins A and E, enhanced egg hatching success, offspring survival to age 15 d, and size of the bursa of Fabricius in offspring. In contrast, repeated maternal GnRH challenges, which elevated yolk testosterone concentrations, enhanced offspring neonatal size, but negatively affected bursa size. However, interaction among the treatments suggests that the positive effect of maternal carotenoid supplementation on plasma bactericidal capacity was mediated by maternal GnRH challenges. Chicks originating from carotenoid‐supplemented hens were less immunosuppressed than those originating from carotenoid‐supplemented + GnRH‐challenged hens, which were less immunosuppressed than chicks from GnRH‐challenged females not supplemented with carotenoids. Females availability of carotenoid enriched diets allows them to enhance the development of offspring immune system via carotenoids and vitamins deposited in egg yolks and offset detrimental effects of androgens deposited by GnRH‐challenged females.     

Maternal condition, yolk androgens and offspring performance: a supplemental feeding experiment in the lesser black-backed gull (Larus fuscus)

It has been proposed that the maternal androgens in avian egg yolk enhance offspring fitness by accelerating growth and improving competitive ability. Because egg quality is strongly influenced by maternal condition, we predicted that females in good condition would produce high-quality eggs with relatively high androgen content. We experimentally enhanced maternal condition by supplementary feeding lesser black-backed gulls (Larus fuscus) during egg formation and compared the concentrations of androstenedione (A4), 5alpha-dihydrotestosterone (DHT) and testosterone (T) in their eggs with those in eggs laid by control females. We also measured circulating levels of T in females immediately after laying. Egg androgens could affect offspring performance directly through chick development and/or indirectly through changes in the competitive ability of a chick relative to its siblings. To avoid confounding these two routes, and to separate effects operating through the egg itself with those operating through experimental changes in parental chick rearing capacity, we fostered eggs from both maternal treatment groups singly into the nests of unmanipulated parents. Contrary to expectation, mothers with experimentally enhanced body condition laid eggs with lower levels of androgens, while exhibiting higher circulating T concentrations post-laying. Despite these lower levels of egg androgen, offspring hatched from eggs laid by mothers in good condition did not show reduced growth or survival when reared in the absence of sibling competition. Our results demonstrate that yolk androgen concentrations vary with the body condition of the female at the time of egg formation and that females in good condition reduced the yolk androgen content of their eggs without altering offspring performance.     

Correlated response in yolk testosterone levels following divergent genetic selection for social behaviour in Japanese quail

Maternal effects are a powerful tool that parents can use to modify the phenotype of their offspring. In birds, the amount of androgens that females deposit in their eggs has been shown to influence early development and adult behavioural phenotypes. Differences in such behavioural strategies have been used as the target of artificial selection programmes with a view to improve animal welfare. In this study, we tested whether artificial selection for divergent social behaviour in Japanese quail had resulted in correlated changes in yolk androgen levels. We used lines that had been selected at the chick stage for high and low motivation to regain contact with a group of conspecific chicks. This procedure has led to important behavioural differences in the high and low line in a suite of behavioural correlates of sociality. We found that eggs laid by the line selected for high motivation for social reinstatement contained more than twice the amount of yolk testosterone of eggs laid by females from the low line, while the unselected line laid eggs with intermediate levels. This finding strongly suggests a functional link between these two traits, and underlines the possible role of yolk androgen modulation in promoting the evolution of behavioural syndromes.     

Hatching asynchrony and maternal androgens in egg yolks of House Wrens

Synchronously and asynchronously hatching clutches of House Wrens Troglodytes aedon usually do not differ in reproductive success. Thus late-hatching nestlings in asynchronously hatching clutches somehow overcome any age- and size-related disadvantages of hatching after their nest-mates. One possible way for them to do this is for female House Wrens to add maternal androgens to the yolk of late-hatching eggs. We tested this hypothesis in a wild population of House Wrens that produces both asynchronously and synchronously hatching clutches. Yolks of eggs from both types of clutches were biopsied and the eggs returned to their nests to hatch. Radioimmunoassays revealed that total androgen levels in the yolk varied within and among clutches. However, total androgen levels in yolks did not vary predictably with egg position in either synchronously or asynchronously hatching clutches. Thus, deposition of androgens in yolk did not follow the expected pattern based on the potential for sibling competition in House Wrens.     

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.     

Do mothers regulate facultative and obligate siblicide by differentially provisioning eggs with hormones?

We examined androgens in clutches of two booby species that differ in their sibling conflict. Blue-footed booby Sula nebouxii chicks show an aggression-submission relationship, aggression is normally moderate and siblicide is facultative. Brown booby Sula leucogaster chicks show an aggression-aggression relationship, aggression of both chicks can be relentless and siblicide is obligate. The parental favoritism hypothesis predicts that egg mass, yolk mass and yolk androgens should decline with laying order less in the blue-footed booby than in the brown booby, to promote the survival of the former's junior chick. The eggs of the blue-footed booby had higher yolk concentrations of 5α-dihydrotestosterone (DHT) and lower concentrations of testosterone (T); both species had similarly high yolk concentrations of androstenedione (A). Intra-clutch variation in yolk DHT, T and A failed to support our predictions. In both species, first and second eggs showed similar concentrations of all three hormones and were of similar size, although in the blue-footed booby (only) yolk masses declined with laying order. Brown booby mothers make junior chicks vulnerable to siblicide by hatching them 5 d after their broodmates, but not by differential allocation of egg androgens or nutrients. Blue-footed booby mothers appear to prepare junior chicks for thriving in a subordinate non-provocative role by hatching them 4 d after their broodmates and providing them with 10% less yolk. To orchestrate agonism within the brood, these boobies may rely more on hatch intervals and yolk provision than on androgens.     

The effects of experimentally manipulated yolk androgens on growth and immune function of male and female nestling collared flycatchers Ficedula albicollis

Hormone-mediated maternal effects may be an important mechanism for adjusting offspring phenotype to particular requirements of the environment. We manipulated the levels of testosterone and androstenedione in the yolk of collared flycatcher Ficedula albicollis eggs to investigate the effects of pre-natal exposure to androgens on growth and immune function. Androgen treatment tended to reduce the growth of males, and enhance the growth of females, as indicated by significant interaction between sex and androgen treatment. Cellular immune function was not affected by androgen treatment or sex. Survival of nestlings until fledging was not related to androgen treatment. Our results indicate that in the collared flycatcher yolk androgens do not involve clear overall benefits during the nestling stage, and that growth-enhancing effects of increased yolk androgen levels on female nestlings are counterbalanced by detrimental effects on male nestlings.     

Negative effects of elevated testosterone on female fecundity in zebra finches

Although factors influencing androgen deposition in the avian egg and its effects on nestling fitness are recently receiving considerable attention, little is known about the potential costs of high testosterone levels in the females. Our study aimed at determining the effect of injections of testosterone (T) in female zebra finches (Taeniopygia guttata), on clutch size, egg mass, yolk mass, and yolk androgen content. Females were given a single bolus injection of T in a range of doses after laying the first egg. Results show that administration of T negatively affected clutch size; the strength of this effect increased with increasing doses of T. Females injected with the highest testosterone dose showed suppressed oviposition of the third and the fourth eggs. Interestingly, testosterone administration made females produce eggs with relatively large yolks, suggesting that T may mediate the trade-off between number and size of eggs. Testosterone injection resulted in elevated levels of androgen in the eggs, in contrast to control clutches, which showed a decreasing pattern of androgen concentration along the laying sequence. We conclude that high androgen investment in eggs may be limited by physiological requirements of the ovulatory process.