Maternal investment during egg laying and offspring sex: an experimental study of zebra finches

To study whether offspring sex is related to the amount of resources invested in eggs we performed an experiment on zebra finches, Taeniopygia guttata. By manipulating their food supply, we forced two groups of females either to increase or to decrease investment in subsequent eggs. Since zebra finches are sexually dimorphic and the reproductive value of the sexes may vary with maternal nutritional status, we predicted that females would adjust the sex of their offspring to egg quality. Females that received poor-quality food for 7 weeks before egg laying, then food supplemented with proteins after they laid the first egg, significantly increased the mass of subsequent eggs. An increase of egg mass with laying sequence was less pronounced in females that received high-quality food before laying and experienced food deterioration after starting to lay. The proportion of sons in subsequent eggs tended to increase in the latter group (although this was marginally significant) but was not related to laying sequence in the other group: these patterns differed significantly between the groups. Offspring sex was not related to egg mass, but newly hatched male chicks were heavier than female chicks. Furthermore, the hatching success of male eggs was lower than that of female eggs. We suggest that differential hatching success of the sexes and sex differences in mass at hatching may constitute important factors shaping brood sex ratios. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Egg size, offspring sex and hatching asynchrony in zebra finches Taeniopygia guttata

Experimental synchronization of onset of incubation was employed in laboratory held zebra finches Taeniopygia guttata to study whether differential resource allocation and possible bias of offspring sex in subsequent eggs in the laying order could mitigate the effects of hatching asynchrony. We found that egg mass increased with laying order, but offspring sex was not related to laying order. Among synchronized clutches, eggs hatched more synchronously than eggs from control nests. Survival probability was related to egg mass, and as expected, this effect differed between experimental groups: it was positive among synchronized broods and not significantly related among asynchronous broods. This suggests that increase in egg mass with the laying order might reduce disparities between early and late hatching chicks. Female nestlings survived better than male nestlings. However their growth was impaired in synchronized broods, whilst growth of males was not affected by hatching synchronization.     

Delayed behavioral effects of postnatal exposure to corticosterone in the zebra finch (Taeniopygia guttata)

Early developmental conditions can significantly influence the growth and survival of many animal species. We studied the consequences of exposure to corticosterone (CORT), a stress hormone, during the nestling stage on two behavioral traits (neophobia, social dominance) measured when the birds had reached independence. Nestling zebra finches (Taeniopygia guttata) were exposed twice daily to exogenous CORT via oral administration for a 12-day period up until fledging. Experimental CORT administration depressed nestling growth rates, confirming results previously obtained in this species. Our data on neophobic behavior revealed a significant interaction between sex and treatment, with CORT-dosed males showing reduced latencies to approach a novel object, while there was little effect of corticosterone treatment on female neophobia. There was no significant effect of age (30 or 50 days), however, there was a non-significant trend towards an interaction between treatment and age, with neophobia increasing with age in the CORT-dosed birds, but decreasing in controls. At 50 days of age previous exposure to corticosterone resulted in reduced success in competitions for a non-food-based resource (a perch) in both sexes. There were no effects of brood size on any behavioral traits measured here, but this may be due to the small range in brood size used. Our results show that elevated levels of stress hormones during postnatal development can have significant effects on important behavioral traits, i.e., neophobia and dominance. Moreover, they confirm the importance of rearing conditions in shaping adult phenotypes.     

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.     

Sex Allocation in Response to Maternal Condition: Different Tactics of Care-Giving by Male and Female Zebra Finches

While behavioral ecologists have studied sex allocation for 30 yr, almost nothing is known about the roles of male vs. female parents in making secondary allocation decisions in species with bi‐parental care. To investigate possible sex differences in tactics in zebra finches, we manipulated the condition of one cohort of females by trimming their flight feathers; a second cohort of females and all males were left intact. Focal observations of feeding behavior revealed that manipulated females did not reduce their feeding time, but rather focused their efforts on rearing sons, while control females allocated effort to rearing daughters. Males mated to manipulated females tended to focus effort to daughters, while control males showed no consistent response. Manipulated females experienced a lower reproductive rate, primarily because of a low daughter rate, relative to control females. Manipulated females also laid smaller clutches and had longer inter‐clutch intervals, which suggest that their ability to metabolize and allocate essential proteins from pectoral muscle mass during the egg‐laying period was impaired. To our knowledge, this study is the first to show that parents of both sexes make active sex allocation decisions when the physiological condition of one parent is manipulated; males do not simply adopt female allocation patterns, but make complementary adjustments. Our results have implications for interpretation of handicapping studies that seek to understand the dynamics of inter‐sexual negotiations of care‐giving. Investigation of sex differences in sex allocation tactics in species with limited dietary protein, such as granivorous birds, is a potentially important direction for future research.     

Tracing the development of learned song preferences in the female zebra finch brain with functional magnetic resonance imaging

In sexually dimorphic zebra finches (Taeniopygia guttata), only males learn to sing their father's song, whereas females learn to recognize the songs of their father or mate but cannot sing themselves. Memory of learned songs is behaviorally expressed in females by preferring familiar songs over unfamiliar ones. Auditory association regions such as the caudomedial mesopallium (CMM; or caudal mesopallium) have been shown to be key nodes in a network that supports preferences for learned songs in adult females. However, much less is known about how song preferences develop during the sensitive period of learning in juvenile female zebra finches. In this study, we used blood-oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to trace the development of a memory-based preference for the father's song in female zebra finches. Using BOLD fMRI, we found that only in adult female zebra finches with a preference for learned song over novel conspecific song, neural selectivity for the father's song was localized in the thalamus (dorsolateral nucleus of the medial thalamus; part of the anterior forebrain pathway, AFP) and in CMM. These brain regions also showed a selective response in juvenile female zebra finches, although activation was less prominent. These data reveal that neural responses in CMM, and perhaps also in the AFP, are shaped during development to support behavioral preferences for learned songs.     

Increased yolk testosterone facilitates prenatal perceptual learning in Northern bobwhite quail (Colinus virginianus)

Prenatal learning plays an important role in the ontogeny of behavior and birds provide a useful model to explore whether and how prenatal exposure to hormones of maternal origin can influence prenatal learning and the development of behavior. In this study we assessed if prenatal exposure to yolk testosterone can influence auditory learning in embryos of Northern bobwhite quail (Colinus virginianus). We experimentally enhanced testosterone concentrations in bobwhite quail eggs prior to incubation. The embryos from these T-treated eggs as well as control embryos that had received the vehicle-only or were non-treated were exposed to an individual bobwhite hen's maternal call for 120 min over the course of the day prior to hatching. All chicks were tested at 24 h following hatching for their auditory preference between the familiar bobwhite maternal call versus an unfamiliar bobwhite maternal call. T-treated chicks spent significantly more time in proximity to the familiar call compared to the unfamiliar call and also showed shorter latencies to approach the familiar call than control birds. Increased emotional reactivity, i.e. propensity to express fear responses, was also found in T-treated chicks. Baseline heart rates recorded in a second group of T-treated embryos and control embryos did not differ, which suggests no effect of yolk testosterone on baseline arousal level. To our knowledge this is the first demonstration of the influence of prenatal exposure to testosterone on auditory learning.     

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.     

Posthatch Oral Estrogen Exposure Impairs Adult Reproductive Performance of Zebra Finch in a Sex-Specific Manner

We determined whether short-term, posthatch oral exposure to estradiol benzoate (EB) or the industrial surfactant octylphenol (OP) could impair the reproductive performance of zebra finches. If so, naturally occurring phytoestrogens and xenoestrogens might influence reproduction in wild populations. Chicks were given oral administration of 10 or 100 nmol EB per gram of body mass (earlier work showed the latter to be the minimum oral dose required to maximally masculinize female song nuclei) or an equimolar amount of OP daily from 5 through 11 days of age. Canola oil was used as a vehicle and control. Reproductive testing was done either in individual pair cages or in communal cages that permitted self-selection of mates, N = 10 pairs per group. Pairs consisted of EB-treated males and females, EB-treated males paired with canola-treated females, vice versa, and canola-treated males and females. Posthatch EB treatment produced sex-specific impairments in reproduction that, in some instances, were additive when both sexes were treated. Egg production was reduced and egg breakage was increased in 100 nmol/g EB-treated male and female pairs. The incidence of missing eggs was increased in 10 nmol/g EB-treated male and female pairs. Candled fertility was reduced in both groups containing 100 nmol/g EB-treated males. The number of hatched chicks was severely reduced in all EB-treated groups. No adverse effects of OP treatment were detected. These significant treatment effects (all P < 0.05) show that posthatch EB treatment profoundly disrupts the reproductive performance of zebra finches, suggesting that exposure to estrogens in the wild could impair the reproductive performance of wild populations.     

Playback of colony sound alters the breeding schedule and clutch size in zebra finch (Taeniopygia guttata) colonies

The hypothesis that social stimulation, derived from the presence and activities of conspecifics, can hasten and synchronize breeding in colonies of birds was tested. A modified playback/recorder system was used to continuously exaggerate the amount of colony sound available to zebra finches throughout their courtship period. Males that heard 'sound supplements' generated from their own colony sang more than males in control colonies that did not receive playback; males that heard samples from a different colony, sang at an intermediate level. Females that were exposed to the vocalizations of their mate and playback from a colony other than their own, laid eggs earlier and more synchronously than females in control colonies. Females that heard the vocalizations of their mate along with playback samples generated from their own colony, laid eggs more synchronously but not earlier than control females. Both acoustic treatments caused females to lay larger clutches. Social stimulation influences the breeding schedule and clutch size in zebra finch colonies. If there are advantages associated with these effects, social stimulation may contribute to the maintenance of colonial breeding systems.     

Sex allocation in response to paternal attractiveness in the zebra finch

Females mated to attractive males are predicted to produce male-biasedbroods. Previous studies on zebra finches, Taeniopygia guttata,in which colored leg rings were used to alter male attractiveness,support this hypothesis. However, because molecular sexing techniqueswere not available, it was not known when during developmentthis bias arose. Also, because both attractive (red-ringed)and unattractive (green-ringed) males were within the same aviary,assortative mating between treatments may have confounded theresults. Using two different experimental designs, we testedwhether the sex ratio of zebra finch eggs and chicks differedin response to paternal ring color whilst controlling for assortativemating between treatments. In the aviary experiment, birds couldinteract socially, but all males in an aviary had the same legring color. In the cage experiment, each female was randomlyassigned a red- or green-ringed mate, thus also eliminatingassortative mating within treatments. Offspring were sexed basedon plumage or using a molecular method. The sex ratio at layingdid not differ between treatments in either the aviary (n =313 eggs) or cage (n = 151 eggs) experiments, suggesting thatfemale zebra finches do not manipulate the primary sex ratioin response to their mate's ring color. However, in the cageexperiment we found greater male embryonic mortality in theattractive group, which resulted in a female-biased sex ratioat sexual maturity, that is, in the opposite direction to thatfound in previous studies. Possible explanations for the disparitybetween our results and those of previous studies are considered.     

Prenatal testosterone treatment alters LH and testosterone responsiveness to GnRH agonist in male sheep

Although evidence is accumulating that prenatal testosterone (T) compromises reproductive function in the female, the effects of excess T in utero on the postnatal development of male reproductive function has not been studied. The aim of this study was to assess the influence of prenatal T excess on age-related changes in pituitary and gonadal responsiveness to GnRH in the male sheep. We used the GnRH agonist, leuprolide (10 microg/kg), as a pharmacologic challenge at 5, 10, 20 and 30 weeks of age. These time points correspond to early and late juvenile periods and the prepubertal and postpubertal periods of sexual development, respectively. LH and T were measured in blood samples collected before and after GnRH agonist administration. The area under the response curve (AUC) of LH increased progressively in both controls and prenatal T-treated males from 5 to 20 weeks of age (P<0.01). The LH responses in prenatal T-treated males were lower at 20 and 30 weeks of age compared to controls (P<0.05). AUC-T increased progressively in control males from 5 through 30 weeks of age and prenatal T-treated males from 5 to 20 weeks of age. The T response in prenatal T-treated males was higher at 20 weeks compared to controls of same age but similar to controls and prenatal T-treated males at 30 weeks of age (P <0.05). Our findings suggest that prenatal T treatment advances the developmental trajectory of gonadal responsiveness to GnRH in male offspring.     

Consequences of prenatal androgen exposure for the reproductive performance of female pheasants (Phasianus colchicus)

Maternal hormones in vertebrate eggs can mediate important forms of maternal effects. However, the function of hormone transfer to the eggs is still debated, especially because long-term fitness consequences have been little studied. We investigated the effect of prenatal exposure to physiologically elevated yolk testosterone (T) levels on reproduction of female pheasants (Phasianus colchicus) in captivity. We found that females hatching from T-injected eggs (T-females) had a lower egg-laying rate than controls, and their eggs were more frequently infertile than those laid by control females. There were no effects of prenatal maternal treatment on egg size and yolk T concentration, but eggs carrying a female embryo laid by T-females had smaller yolks than eggs with a male embryo, while there was no sex difference in yolk size among the eggs laid by control females. Progeny sex ratio was unaffected by maternal treatment. These findings suggest that the transfer of high androgen levels to the eggs by the mother is constrained by complex trade-offs between direct effects on her daughters' reproduction and by trans-generational differential consequences on male and female descendants.     

Testosterone metabolism in the avian hypothalamus

Many central actions of testosterone (T) require the transformation of T into several metabolites including 5-dihydrotestosterone (5-DHT) and estradiol (E₂). In birds as in mammals, 5-DHT and E₂, alone or in combination, mimic most behavioral effects of T. The avian brain is, in addition, able to transform T into 5β-DHT, a metabolite which seems to be devoid of any behavioral or physiological effects, at least in the context of reproduction. By in vitro product-formation assays, we have analyzed the distribution, sex differences and regulation by steroids of the 3 main T metabolizing enzymes (aromatase, 5- and 5β-reductases) in the brain of the Japanese quail (Coturnix c. japonica) and the zebra finch (Taeniopygia guttata castanotis). In the hypothalamus of quail and finches, aromatose activity is higher in males than in females. It is also decreased by castration and increased by T. The activity of the 5-reductase is not sexually differentiated nor controlled by T. The 5β-reductase activity is often higher in females than in males but this difference disappears in gonadectomized birds and no clear effect of T can be observed at this level. The zebra finch brain also contains a number of steroid-sensitive telencephalic nuclei [e.g. hyperstriatum ventrale, pars caudale (HVc) and robustus archistriatalis (RA)] which play a key role in the control of vocalizations. These nuclei also contain T-metabolizing enzymes but the regulation of their activity is substantially different from what has been observed in the hypothalamus. Aromatase activity is for example higher in females than in males in HVc and RA and the enzyme in these nuclei is not affected by castration nor T treatment. In these nuclei, the 5-reductase activity is higher in males than in females and the reverse is true for the 5β-reductase. These sex differences in activity are not sensitive to gonadectomy and T treatment and might therefore be organized by neonatal steroids. We have been recently able to localize aromatase-immunoreactive (AR-ir) neurons by ICC in the brain of the quail and zebra finch. Positive cells are found in the preoptic area, ventromedial and tuberal hypothalamus. AR-ir material is found in the perikarya of cells and fills the entire cellular processes including axons. At the electron microscope level, immunoreactive material can clearly be observed in the synaptic boutons. This observation raises questions concerning the mode of action of estrogens produced by central aromatization of T.     

Paradoxical hypermasculinization of the zebra finch song system by an antiestrogen

Exogenous estrogens, when administered to hatchling female zebra finches, masculinize the morphology and function of their neural vocal control system. The first of two experiments evaluated whether tamoxifen citrate is an antiestrogen in zebra finches, and the second determined whether it would block the masculinization hypothesized to be caused in hatchling males by the males' endogenous estradiol. In the first experiment adult female zebra finches were ovariectomized and injected for 10 days with estradiol benzoate (EB), tamoxifen, EB and tamoxifen combined, or vehicle (control). The dependent variable was oviduct weight. The EB-stimulated growth of the oviduct was blocked by tamoxifen, which had no effects when administered alone. Thus, tamoxifen acts as an antiestrogen in the zebra finch oviduct. In Experiment 2, male and female zebra finches were treated with tamoxifen or vehicle for the first 20 days after hatching. The males were castrated at 20 days. At 60 days we compared the song control regions of experimental and control males and females. In both sexes tamoxifen increased the somatic areas of neurons in RA (robust nucleus of the archistriatum), HVc (caudal nucleus of the ventral hyperstriatum), and MAN (magnocellular nucleus of the anterior neostriatum). Tamoxifen also increased the volumes of HVc, RA, MAN, and Area X in males. Thus, tamoxifen failed to block masculinization of males, but masculinized females and hypermasculinized males. Tamoxifen's hypermasculinization of the male and masculinization of the female song system is paradoxical given that (1) estradiol does not have similar effects on the male song system, and (2) tamoxifen antagonizes the effects of EB in the oviduct.     

Nest destruction elicits indiscriminate con‐ versus heterospecific brood parasitism in a captive bird

Following nest destruction, the laying of physiologically committed eggs (eggs that are ovulated, yolked, and making their way through the oviduct) in the nests of other birds is considered a viable pathway for the evolution of obligate interspecific brood parasitism. While intraspecific brood parasitism in response to nest predation has been experimentally demonstrated, this pathway has yet to be evaluated in an interspecific context. We studied patterns of egg laying following experimental nest destruction in captive zebra finches, Taeniopygia guttata, a frequent intraspecific brood parasite. We found that zebra finches laid physiologically committed eggs indiscriminately between nests containing conspecific eggs and nests containing heterospecific eggs (of Bengalese finches, Lonchura striata vars. domestica), despite the con‐ and heterospecific eggs differing in both size and coloration. This is the first experimental evidence that nest destruction may provide a pathway for the evolution of interspecific brood parasitism in birds.     

Anatomically Discrete Sex Differences in Neuroplasticity in Zebra Finches as Reflected by Perineuronal Nets

Large morphological sex differences in the vertebrate brain were initially identified in song control nuclei of oscines. Besides gross differences between volumes of nuclei in males and females, sex differences also concern the size and dendritic arborization of neurons and various neurochemical markers, such as the calcium-binding protein parvalbumin (PV). Perineuronal nets (PNN) of the extracellular matrix are aggregates of different compounds, mainly chondroitin sulfate proteoglycans, that surround subsets of neurons, often expressing PV. PNN develop in zebra finches song control nuclei around the end of the sensitive period for song learning and tutor deprivation, known to delay the end of the song learning sensitive period, decreases the numbers of PNN in HVC. We demonstrate here the existence in zebra finches of a major sex difference (males > females) affecting the number of PNN (especially those surrounding PV-positive cells) in HVC and to a smaller extent the robust nucleus of the arcopallium, RA, the two main nuclei controlling song production. These differences were not present in Area X and LMAN, the lateral magnocellular nucleus of the anterior nidopallium. A dense expression of material immunoreactive for chondroitin sulfate was also detected in several nuclei of the auditory and visual pathways. This material was often organized in perineuronal rings but quantification of these PNN did not reveal any sex difference with the exception that the percentage of PNN surrounding PV-ir cells in the dorsal lateral mesencephalic nucleus, MLd, was larger in females than in males, a sex difference in the opposite direction compared to what is seen in HVC and RA. These data confirm and extend previous studies demonstrating the sex difference affecting PNN in HVC-RA by showing that this sex difference is anatomically specific and does not concern visual or auditory pathways.     

Compensatory investment in zebra finches: females lay larger eggs when paired to sexually unattractive males

The classical version of the differential allocation hypothesis states that, when females reproduce over their lifetime with partners that differ in their genetic quality, they should invest more in reproduction with high-quality males. However, in species with lifetime monogamy, such as the zebra finch, partner quality will typically remain the same. In this case, the compensatory investment (CI) hypothesis predicts higher investment for low-quality males, because low genetic quality offspring are more dependent on maternal resources. Here, we show that female zebra finches invested more resources, both in terms of egg volume and yolk carotenoid content, when paired to a low genetic quality male, as judged from his previous ability to obtain extra-pair paternity in aviary colonies. We also found that females deposited slightly larger amounts of testosterone into eggs when paired to a low parental quality male, as judging from his previous success in rearing offspring. This is, to our knowledge, the first experimental support for the CI hypothesis in a species with lifetime monogamy. We stress that in more promiscuous species, the benefits of classical differential allocation may partly be neutralized by the supposed benefits of CI.     

Sex differences in the telencephalic song control circuitry in Bengalese finches (Lonchura striata var. domestica)

Bengalese finches, Lonchura striata, are extremely sexually dimorphic in their singing behavior; males sing complex songs, whereas females do not sing at all. This study describes the developmental differentiation of the brain song system in Bengalese finches. Nissl staining was used to measure the volumes of four telencephalic song nuclei: Area X, HVC, the robust nucleus of the arcopallium (RA), and the lateral portion of the magnocellular nucleus of the anterior nidopallium (LMAN). In juveniles (circa 35 days old), Area X and the HVC were well developed in males, while they were absent or not discernable in females. The RA was much larger in males but barely discernable in females. In males, the volumes of Area X and the RA increased further into adulthood, but that of the HVC remained unchanged. The LMAN volume was greater in juveniles than in adults, and there was no difference in the LMAN volume between the sexes. The overall tendency was similar to that described in zebra finches, except for the volume of the RA, where the degree of sexual dimorphism is larger and the timing of differentiation occurs earlier in Bengalese finches. Motor learning of the song continues until day 90 in zebra finches, but up to day 120 in Bengalese finches. Earlier neural differentiation and a longer learning period in Bengalese finches compared with zebra finches may be related to the more elaborate song structures of Bengalese finches.     

Social mediation of vocal amplitude in a songbird, Taeniopygia guttata

Bird vocalizations are produced under various social contexts. It could therefore benefit birds, as social contexts change, to alter the amplitude of their signals. We tested this by recording male and female zebra finches, Taeniopygia guttata, when they were placed in isolation, in auditory contact, and in visual and auditory contact with each sex. The majority of females increased call amplitude only when in auditory contact with either sex. Most males increased both song and call amplitudes only when in visual and auditory contact with either sex. We found no changes in patterns of harmonic suppression (timbre) for males or females across social conditions. One explanation for the sex difference is that females increase amplitude for affiliative reasons, whereas males increase amplitude to advertise fitness.