A nonsemen copulatory fluid influences the outcome of sperm competition in Japanese quail.

Sperm competition is a powerful and widespread evolutionary force that drives the divergence of behavioural, physiological and morphological traits. Elucidating the mechanisms governing differential fertilization success is a fundamental question of sperm competition. Both sperm and nonsperm ejaculate components can influence sperm competition outcomes. Here, we investigate the role of a nonsemen copulatory fluid in sperm competition. Male Japanese quail possess a gland that makes meringue-like foam. Males produce and store foam independent of sperm and seminal fluid, yet transfer foam to females during copulation. We tested whether foam influenced the outcome of sperm competition by varying foam state and mating order in competitive matings. We found that the presence of foam from one male decreased the relative fertilization success of a rival, and that foam from a given male increased the probability he obtained any fertilizations. Mating order also affected competitive success. Males mated first fertilized proportionally more eggs in a clutch and had more matings with any fertilizations than subsequent males. We conclude that the function of foam in sperm competition is mediated through the positive interaction of foam with a male's sperm, and we speculate whether the benefit is achieved through improving sperm storage, fertilizing efficiency or retention. Our results suggest males can evolve complex strategies to gain fertilizations at the expense of rivals as foam, a copulatory fluid not required for fertilization, nevertheless, has important effects on reproductive performance under competition.

Maternally derived carotenoid pigments affect offspring survival, sex ratio, and sexual attractiveness in a colorful songbird.

In egg-laying animals, mothers can influence the development of their offspring via the suite of biochemicals they incorporate into the nourishing yolk (e.g. lipids, hormones). However, the long-lasting fitness consequences of this early nutritional environment have often proved elusive. Here, we show that the colorful carotenoid pigments that female zebra finches (Taeniopygia guttata) deposit into egg yolks influence embryonic and nestling survival, the sex ratio of fledged offspring, and the eventual ornamental coloration displayed by their offspring as adults. Mothers experimentally supplemented with dietary carotenoids prior to egg-laying incorporated more carotenoids into eggs, which, due to the antioxidant activity of carotenoids, rendered their embryos less susceptible to free-radical attack during development. These eggs were subsequently more likely to hatch, fledge offspring, produce more sons than daughters, and produce sons who exhibited more brightly colored carotenoid-based beak pigmentation. Provisioned mothers also acquired more colorful beaks, which directly predicted levels of carotenoids found in eggs, thus indicating that these pigments may function not only as physiological 'damage-protectants' in adults and offspring but also as morphological signals of maternal reproductive capabilities.

Anhydrolutein in the zebra finch: a new, metabolically derived carotenoid in birds.

Many birds acquire carotenoid pigments from the diet that they deposit into feathers and bare parts to develop extravagant sexual coloration. Although biologists have shown interest in both the mechanisms and function of these colorful displays, the carotenoids ingested and processed by these birds are poorly described. Here we document the carotenoid-pigment profile in the diet, blood and tissue of captive male and female zebra finches (Taeniopygia guttata). Dietary carotenoids including: lutein; zeaxanthin; and beta-cryptoxanthin were also present in the plasma, liver, adipose tissue and egg-yolk. These were accompanied in the blood and tissues by a fourth pigment, 2',3'-anhydrolutein, that was absent from the diet. To our knowledge, this is the first reported documentation of anhydrolutein in any avian species; among animals, it has been previously described only in human skin and serum and in fish liver. We also identified anhydrolutein in the plasma of two closely related estrildid finch species (Estrilda astrild and Sporaeginthus subflavus). Anhydrolutein was the major carotenoid found in zebra finch serum and liver, but did not exceed the concentration of lutein and zeaxanthin in adipose tissue or egg yolk. Whereas the percent composition of zeaxanthin and beta-cryptoxanthin were similar between diet and plasma, lutein was comparatively less abundant in plasma than in the diet. Lutein also was proportionally deficient in plasma from birds that circulated a higher percentage of anhydrolutein. These results suggest that zebra finches metabolically derive anhydrolutein from dietary sources of lutein. The production site and physiological function of anhydrolutein have yet to be determined.

Masculinized sexual partner preference in female zebra finches with sex-reversed gonads.

Previous research in the zebra finch, a socially monogamous pair-bonding species, suggests that the preference for opposite-sex partners may arise in part through the organizing actions of sex steroids. To further investigate this process, zebra finch eggs were injected with 20 microg fadrozole, a potent estrogen synthesis inhibitor, or with the saline vehicle on embryonic day 5. As adults they were given two-choice sexual partner preference tests followed by group aviary tests. Fadrozole females had masculinized beak color and had testes or ovotestes instead of ovaries. Males were not affected by fadrozole; they did not differ from controls on any measure. In contrast, sexual partner preference was substantially masculinized in fadrozole females in the group aviary tests. Untreated males given a choice between fadrozole and untreated females preferred the untreated females, but this was equally the case when they were given a choice between saline-treated and untreated females. These results suggest that males do not specifically avoid females with testes and that male avoidance is unlikely to explain why fadrozole-treated females pair with other females. The present data add to the evidence that actions of gonadal steroids during development contribute to adult sex differences in partner preference in this pair-bonding species. Furthermore, because fadrozole-treated females do not produce audible song, the mechanisms regulating partner preference and song system development are dissociated.

Removal of adult males from the rearing environment increases preference for same-sex partners in the zebra finch.

The developmental processes producing preferences for opposite-sex mating partners are not well understood. Zebra finches, Taeniopygia guttata, are colonial and socially monogamous with biparental care. To determine whether the early social environment contributes to sexual partner preference, we removed adult males from breeding colonies when the oldest chicks were less than 1 week old (male-removal rearing) or left them in the breeding cage (control rearing). At independence, male-removal and control offspring were moved to unisex cages. As adults they were given two-choice tests with male versus female stimuli followed by group aviary tests. Male-removal subjects, unlike controls, did not prefer opposite-sex stimuli in the two-choice tests. Male-removal subjects were less likely than controls to successfully pair with opposite-sex birds in the group aviary tests; 38% of them paired with a same-sex bird. Thus early social experience may contribute to a critical component of mate choice, choosing the opposite sex, in this pair-bonding species. Copyright 2000 The Association for the Study of Animal Behaviour.

Effect of septal lesions on male song and aggression in the colonial zebra finch (Taeniopygia guttata) and the territorial field sparrow (Spizella pusilla)

The present investigation assessed the effect of lesions of the septum on male courtship and aggression in the territorial field sparrow (Spizella pusilla) and the colonial zebra finch (Taeniopygia guttata). In addition, pair-bonding and a variety of other social behaviors were examined in the zebra finch and dawn song (both the strictly agonistic song type and the multipurpose song type) was examined in the field sparrow. Zebra finches were tested in three phases both before and after receiving bilateral electrolytic lesions of the septum or sham surgery. These phases were: (1) competition tests in which a subject and a stimulus male were exposed to a female in an adjacent cage; (2) sexual behavior tests with a female; and (3) 10-day group cage tests in which subjects were in a mixed-sex environment. Aggressive behaviors (chases, threats, beak fences and pecks) were significantly reduced by septal lesions but not by sham surgery. Directed song (courtship) was significantly reduced in sexual behavior tests, with similar trends in other testing phases. Male field sparrows were tested 2 days pre-surgery and 2 days post-surgery in outdoor aviaries placed in their natural habitat. Tests consisted of dawn song observations and observations of courtship and aggression following introduction of a female to the subject's aviary, which was followed 10 min later by the introduction of another male (without removing the female). Septal lesions significantly facilitated both overt aggression (chases) and the number of simple (multi-purpose) songs. These results provide evidence that the septum participates in the regulation of male aggression and song in songbirds, and further suggest that variations in septal function may exist between territorial and colonial species.

Effect of septal lesions on male song and aggression in the colonial zebra finch (Taeniopygia guttata) and the territorial field sparrow (Spizella pusilla)

The present investigation assessed the effect of lesions of the septum on male courtship and aggression in the territorial field sparrow (Spizella pusilla) and the colonial zebra finch (Taeniopygia guttata). In addition, pair-bonding and a variety of other social behaviors were examined in the zebra finch and dawn song (both the strictly agonistic song type and the multipurpose song type) was examined in the field sparrow. Zebra finches were tested in three phases both before and after receiving bilateral electrolytic lesions of the septum or sham surgery. These phases were: (1) competition tests in which a subject and a stimulus male were exposed to a female in an adjacent cage; (2) sexual behavior tests with a female; and (3) 10-day group cage tests in which subjects were in a mixed-sex environment. Aggressive behaviors (chases, threats, beak fences and pecks) were significantly reduced by septal lesions but not by sham surgery. Directed song (courtship) was significantly reduced in sexual behavior tests, with similar trends in other testing phases. Male field sparrows were tested 2 days pre-surgery and 2 days post-surgery in outdoor aviaries placed in their natural habitat. Tests consisted of dawn song observations and observations of courtship and aggression following introduction of a female to the subject's aviary, which was followed 10 min later by the introduction of another male (without removing the female). Septal lesions significantly facilitated both overt aggression (chases) and the number of simple (multi-purpose) songs. These results provide evidence that the septum participates in the regulation of male aggression and song in songbirds, and further suggest that variations in septal function may exist between territorial and colonial species.

Testosterone increases singing and aggression but not male-typical sexual partner preference in early estrogen treated female zebra finches.

Female zebra finches given estradiol benzoate (EB) as nestlings and testosterone propionate (TP) as adults show masculinized sexual partner preference, preferring females instead of males. This suggests an organizational effect of EB on sexual partner preference in a socially monogamous species that pairs for life. It is not known whether there is an activational hormone effect on sexual partner preference in this species, or whether adult testosterone treatment is necessary for masculinized preference to be expressed. In this experiment females were injected with EB daily for the first 2 weeks posthatching. As adults they were given TP filled or empty implants. Subjects were then given two-choice preference tests with male vs female stimuli, in which singing as well as proximity to the stimuli was recorded, followed by tests in a group aviary for social behavior and pairing preference. Females with TP implants sang more than females with empty implants and were more aggressive toward other females. They did not, however, differ from females with empty implants in any measure of sexual partner preference. Neither group showed a marked preference for males; instead both groups were equally interested in males and females. Thus adult testosterone treatment is not necessary for early estrogen treated females to show a shift in sexual partner preference in the male-typical direction.

Effect of intraseptal vasotocin and vasoactive intestinal polypeptide infusions on courtship song and aggression in the male zebra finch (Taeniopygia guttata).

The present experiments were conducted to test the hypothesis that septal arginine vasotocin (AVT) and vasoactive intestinal polypeptide (VIP) modulate directed song (a courtship behaviour) and aggression in male zebra finches (Taeniopygia guttata). Subjects were surgically fitted with a guide cannula directed at the septum. Following recovery they were tested for aggression and directed song following infusions of AVT, its antagonist (anti-vasopressin, AVP), and saline volume control. Infusion of the AVT antagonist significantly reduced all three aggressive behaviours measured (pecks, beak fences and chases); and AVT infusion significantly facilitated beak fencing. Vasoactive intestinal polypeptide treatment significantly reduced pecking. No treatment produced a change in directed song. Comparison with findings in mammals suggests that modulation of aggression by septal AVT (or AVP) is evolutionarily conserved in vertebrates, but modulation of aggression by VIP has not previously been reported for any vertebrate.

The foam production system of the male Japanese quail: characterization of structure and function.

The research described here characterizes a unique neuromuscular system involved in reproductive behavior--the foam production system of the male Japanese quail (Coturnix japonica). Male quail produce a large amount of foam that is transferred to the female during copulation, enhancing male fertilization success. The source is the foam gland complex, a large sexually dimorphic, androgen sensitive, external protuberance of the dorsal cloaca, consisting of glandular units interdigitated with striated muscle fibers of the sphincter cloacae muscle (mSC). Electromyographic (EMG) analysis of mSC activity in freely moving males interacting with females revealed different characteristics of the EMG signal during copulation, voiding of excreta, and other mSC movement. The amount of mSC activity and also the amount of foam produced were greatly increased by the presence of a female behind a screen. Denervation of mSC eliminated normal mSC movement and also abolished foam production, confirming that mSC activity is the mechanism for foam production. The spinal cord locations of the motoneurons innervating the major cloacal muscles, including mSC, were determined by injecting cholera-toxin conjugated horseradish peroxidase into each muscle. Labelled somata with multiple primary dendrites were located in Area IX of the lateral motor column of synsacral segments 7, 8, or 9 or 8, 9, and 10. The motoneurons serving mSC were intermingled with those projecting to the other cloacal muscles, but there were differences in the rostralcaudal placement of these neural populations. Thus mSC activity is an integral part of the male's reproductive behavior, mSC activity can be socially stimulated, and mSC activity occurring in anticipation of copulation is likely to be functionally significant. Continued investigation of this highly accessible system has the potential to shed light on the mechanisms by which complex motor acts are produced and hormonally regulated.

Role of the archistriatal nucleus taeniae in the sexual behavior of male Japanese quail (Coturnix japonica): a comparison of function with the medial nucleus of the amygdala in mammals.

Nucleus taeniae (Tn) is a prominent cell group within the medial archistriatum of birds. Based upon similarities in sex-steroid binding sites, this nucleus has been hypothesized to be homologous to the medial nucleus of the amygdala (Me) in mammals, which is known to modulate the expression of sexual behavior in rodents. We therefore tested whether or not Tn likewise plays a role in the expression of sexual behavior in male Japanese quail. We found that bilateral damage to Tn produced deficits in several components of male responses toward female stimuli that were indicative of decreased sexual arousal, including goal-oriented responses, vocalizations associated with courtship, and motor reflexes that precede copulation. Our results suggest that Tn influences a wide range of behavioral functions in response to sexual stimuli, and they indicate a function for this nucleus similar to that subserved by the Me in mammals. These results strengthen the argument that these sex-steroid accumulating cell groups are homologous and suggest a conservation of function for them despite the vastly divergent evolutionary histories separating birds and mammals.

Sensitivity and behavioral responses to the pheromone androstenone are not mediated by the vomeronasal organ in domestic pigs.

Based largely on results of studies of laboratory rodents, the vomeronasal or accessory olfactory system is believed to function mainly in social communication, mediating the effects of stimuli such as urine or glandular secretions on the behavior or endocrine response of conspecifics. In the domestic pig (Sus scrofa), the steroid androstenone has been identified as a pheromone that facilitates expression of both attraction to the male and a receptive mating stance in estrous females. Though the domestic pig is one of the few vertebrate species in which the identity of a compound that functions as a pheromone is known, the role of the vomeronasal system in domestic pigs has never been investigated. We have examined the role of the vomeronasal organ in mediating the pheromonal effects of androstenone in pigs. In addition, we have examined the structure of the vomeronasal organ at the gross and light-microscopic levels. The vomeronasal organ appears functional, with sensory epithelium lining the medial wall, and has access to stimuli from both the oral and nasal cavities. To determine whether the vomeronasal organ is necessary for androstenone detection or attraction or receptive behavior in female pigs, access to the vomeronasal organ was blocked with surgical cement, and androstenone detection threshold and sexual behavior were measured. Experimental animals did not differ significantly in androstenone sensitivity, measured behaviorally, from untreated controls. Vomeronasal organ-blocked animals also did not differ from untreated controls in either androstenone-mediated receptive standing behavior or attraction to the odor of androstenone. We conclude that in the domestic pig, the vomeronasal organ is not necessary for androstenone detection or androstenone-mediated sexual behavior in estrous females.

Organizational actions of sex hormones on sexual partner preference.

Sexual dimorphism in copulatory behavior results from organizational actions of sex steroids (permanent effects of sex steroids occurring during early development). Reproductive success depends not only on copulatory behavior, but also on mate choice, which is often sexually dimorphic as well. The clearest example is sexual partner preference: the preference of males for female sexual partners and females for males. Are organizational hormone actions responsible for sexual differentiation of sexual partner preference? The zebra finch (Taeniopygia guttata) is a potentially valuable species for addressing this question, because the birds form life-long socially monogamous pair bonds. In one experiment, both early estrogen treatment (injection with estradiol benzoate-EB-for the first 2 weeks posthatch) and unisex housing during juvenile development independently resulted in a preference for females over males in two-choice tests, and only females that experienced both EB treatment and unisex living were more likely than controls to pair with other females in colony tests. In a second experiment, females injected with an estrogen synthesis inhibitor for the first week posthatch preferred to spend time near females instead of males in two-choice tests, unlike control females. These experiments suggest that sexual partner preference may result from organizational hormone actions in this pair-bonding species. Possible neural mechanisms or sites that could underly hormonal organization of sexual partner preference in birds and mammals include the anterior hypothalamic/preoptic area, the corticomedial amygdala, and its avian homologue nucleus taeniae of the archistriatum, the septum, and peripheral sensory processes.

Sexual partner preference in female zebra finches: the role of early hormones and social environment.

This experiment investigated the effects of early estrogen treatment and sex composition of the social environment on sexual partner preference in female zebra finches, a pair-bonding socially monogamous species. Birds were injected daily with estradiol benzoate (EB) or the steroid vehicle for the first 2 weeks posthatch and then lived in either a unisex (all-female) or a mixed sex group from 40 to 100 days. After 100 days birds were implanted with testosterone propionate and given three kinds of tests: tests with a stimulus female, two-choice mate preference tests with male and female stimuli, and colony tests to assess pairing preference in a more naturalistic context. Both EB and unisex housing independently resulted in a preference for females (masculinized preference) in the two-choice tests, but only females with both EB treatment and unisex living were more likely to pair with females in the colony tests. Sexual partner preference, a key sexually dimorphic component of mate choice, appears to be organized by sex steroids in this pair-bonding species, but in a manner that may be mediated by the social environment.

Olfactory sensitivity to the pheromone, androstenone, is sexually dimorphic in the pig.

Sexually dimorphic pheromone pathways have been used successfully to study insect olfactory coding. As one of the few mammalian species with an identified sex pheromone, the domestic pig (Sus scrofa) may be an ideal vertebrate species in which to examine sex differences in olfactory processing of a specific stimulus. In this experiment, androstenone and control odor detection thresholds were measured in adult male, female, and castrated male pigs. In an operant task, pigs were tested with descending concentration series of both androstenone and geraniol. All groups were equally sensitive to geraniol, but there was a sex difference in sensitivity to the odor of androstenone. Female pigs' detection threshold was a dilution fivefold lower than the threshold for intact males. Castrated males did not differ significantly from either males or females. This is the first example of a sexual dimorphism in sensitivity to a mammalian pheromone.

Photoperiod affects the morphology of a sexually dimorphic nucleus within the preoptic area of male Japanese quail.

Changes in the gross and cellular morphology of the nucleus preopticus medianus (POMn) were measured in response to changes in photoperiod in adult male Japanese quail (Coturnix japonica). POMn volume and the soma size of a dorsolateral population of neurons within POMn decreased when birds were moved from long day housing conditions (16L,8D) to short day housing conditions (8L,16D), and then increased again when birds were moved back to long day conditions, presumably as a function of the changes in circulating testosterone that accompanied changing daylengths. Male Japanese quail exhibit sexual behavior only when housed under long day housing conditions that approximate the photoperiod of the spring/summer breeding season, and do not exhibit sexual behavior when housed under short day conditions characteristic of fall/winter. Because POMn is known to be critically involved in the expression of male copulatory behavior, these morphological changes in the adult brain likely represent key functional events associated with the seasonal regulation of sexual behavior in male Japanese quail.

Sexual differentiation of brain and behavior in the zebra finch: critical periods for effects of early estrogen treatment.

In order to determine the critical period(s) during which estrogen alters sexually dimorphic behavior and neuroanatomy in zebra finches (Poephila guttata), nestlings were injected daily with 20 micrograms estradiol benzoate (EB) during posthatching week 1, week 2, week 3, or weeks 1, 2, and 3. At 7 months of age, birds were implanted with testosterone propionate and tested with female partners for singing, dancing, and copulatory mounting. Brains were subsequently processed for morphometry, and the volumes of the song system nuclei HVC, area X, and RA and the soma sizes and densities of neurons in RA were determined. Males given EB during week 1 failed to mount. Females given EB during week 1 were fully masculinized with respect to dancing and RA neuron soma size and density, and were partially masculinized with respect to song nuclei volumes and singing. Treatment beginning after week 1 was ineffective or less effective for all measures. Only for RA neuron measures was treatment for all three weeks more effective than week 1 treatment. Thus the first post-hatching week is the most influential period of those tested for effects of exogenous estrogen on sexual differentiation in this species, and is a period during which both masculinization of females and demasculinization of males is possible.

Ontogeny of a sexually dimorphic nucleus in the preoptic area of the Japanese quail (Coturnix japonica).

The nucleus preopticus medianus (POMn) is a sexually dimorphic nucleus in Japanese quail (Coturnix japonica) that is critically involved in the hormonal activation of male copulatory behavior. The larger volume apparent in males appears to depend upon circulating testosterone [Brain Res., 416 (1987) 59-68; J. Comp. Neurol., 303 (1991) 443-456]. The present study determined when during normal development this nucleus becomes dimorphic. POMn and a control nucleus, the nucleus commissurae pallii (nCPa), were traced from Nissl-stained coronal sections (40 microns) from animals sacrificed at 2, 3, 4, 5, 6 or 7 weeks of age. Areas were measured and used to calculate volume. POMn volumes were not significantly different in males and females through 5 weeks of age. The dimorphism in POMn volume then became apparent at 6 weeks of age as a function of an increase in male POMn volume between 5 and 6 weeks of age. No significant differences were apparent at any developmental age in nCPa volume. The appearance of a sexual dimorphism in POMn volume is coincident with the pubertal surge in testosterone that occurs between 5 and 6 weeks of age [Horm. Behav., 11 (1978) 175-182], and is also coincident with behavioral sexual maturity.

Sexual dimorphism in the avian brain is not limited to the song system of songbirds: a morphometric analysis of the brain of the quail (Coturnix japonica).

Nissl-stained sections of adult male and female quail brains were examined for possible sex differences in the sizes of nuclei implicated in reproductive function or known to be dimorphic in songbirds, including nucleus magnocellularis preopticus, two additional preoptic nuclei, n. ventromedialis hypothalami, n. taeniae, n. hypoglossus and n. commissurae pallii. There was a significant sex difference in the volume of one of the preoptic nuclei (which was 38% larger in males), known to be a critical area for the hormonal activation of male copulatory behavior, which cannot be accounted for by overall brain size dimorphism. It is suggested that this nucleus be named nucleus preopticus medianus (POMn). There was no sex difference in the average neuronal density or cross-sectional soma area within POMn; rather, the male POMn consists of a greater total number of neurons than the female POMn. No other nuclei that were measured were dimorphic in volume, including n. hypoglossus. There was small but significant lateral asymmetry in n. commissurae pallii, the left side being about 10% larger in volume.