In many cooperatively breeding species non-breeding individuals help to rear the offspring of breeders. The physiological mechanisms that regulate such cooperative helping behavior are poorly understood, but may have been co-opted, during the evolution of cooperative breeding, from pre-existing mechanisms that regulated parental care. Key among these may be a role for prolactin. Here we investigate whether natural variation in circulating prolactin levels predicts both parental and helper contributions to nestling provisioning in wild cooperatively breeding white-browed sparrow weavers, Plocepasser mahali. In sparrow weaver groups, a dominant pair monopolize reproduction and non-breeding subordinates help with nestling feeding. We show that: (i) among parents, females feed their nestlings at higher rates, make longer provisioning visits, and have higher prolactin levels than males; and (ii) among subordinates, engaged in cooperative helping behavior, those within their natal groups feed nestlings at higher rates and have higher prolactin levels than immigrants. Accordingly, continuous variation in prolactin levels positively predicts nestling provisioning rates and provisioning visit durations when all bird classes are combined. These relationships are principally driven by differences among bird classes; the more limited within-class variation in prolactin and provisioning traits were not evidently correlated, for which a range of potential explanations exist. While these correlative findings are broadly consistent with the hypothesis that parental care and cooperative helping behavior are regulated by a shared mechanism, experiments are now needed to establish whether prolactin plays a causal role, and if so, whether it regulates continuous variation in provisioning rate or simply activates provisioning behavior.

Increased thyrotrophin-stimulating hormone (TSH) expression in the pars tuberalis is assumed to be an early step in the neuroendocrine mechanism transducing photoperiodic information. The present study aimed to determine the relationship between long-photoperiod (LP) and diurnal TSH gene expression in the juvenile chicken by comparing LP-photostimulated birds with groups kept on a short photoperiod (SP) for 1 or 12days. TSH expression increased by 3- and 23-fold after 1 and 12days of LP-photostimulation both during the day and at night. Under both SP and LP conditions, TSH expression was between 3- and 14-fold higher at night than in the day, suggesting that TSH expression cycles in a diurnal pattern irrespective of photoperiod. The ratio of DIO2/3 was decreased on LPs, consequent to changes in DIO3 expression, although there was no evidence of any diurnal effect on DIO2 or DIO3 expression. Plasma prolactin concentrations revealed both an effect of LPs and time-of-day. Thus, TSH expression changes in a dynamic fashion both diurnally and in response to photoperiod.

Background A major step towards the success of chickens as a domesticated species was the separation between maternal care and reproduction. Artificial incubation replaced the natural maternal behaviour of incubation and, thus, in certain breeds, it became possible to breed chickens with persistent egg production and no incubation behaviour; a typical example is the White Leghorn strain. Conversely, some strains, such as the Silkie breed, are prized for their maternal behaviour and their willingness to incubate eggs. This is often colloquially known as broodiness. Results Using an F 2 linkage mapping approach and a cross between White Leghorn and Silkie chicken breeds, we have mapped, for the first time, genetic loci that affect maternal behaviour on chromosomes 1, 5, 8, 13, 18 and 19 and linkage group E22C19W28. Paradoxically, heterozygous and White Leghorn homozygous genotypes were associated with an increased incidence of incubation behaviour, which exceeded that of the Silkie homozygotes for most loci. In such cases, it is likely that the loci involved are associated with increased egg production. Increased egg production increases the probability of incubation behaviour occurring because egg laying must precede incubation. For the loci on chromosomes 8 and 1, alleles from the Silkie breed promote incubation behaviour and influence maternal behaviour (these explain 12 and 26 % of the phenotypic difference between the two founder breeds, respectively). Conclusions The over-dominant locus on chromosome 5 coincides with the strongest selective sweep reported in chickens and together with the loci on chromosomes 1 and 8, they include genes of the thyrotrophic axis. This suggests that thyroid hormones may play a critical role in the loss of incubation behaviour and the improved egg laying behaviour of the White Leghorn breed. Our findings support the view that loss of maternal incubation behaviour in the White Leghorn breed is the result of selection for fertility and egg laying persistency and against maternal incubation behaviour.

Clutch size is a key avian fitness and life history trait. A physiological model for clutch size determination (CSD), involving an anti-gonadal effect of prolactin (PRL) via suppression of luteinizing hormone (LH), was proposed over 20 years ago, but has received scant experimental attention since. The few studies looking at a PRL-based mechanistic hypothesis for CSD have been equivocal, but recent experiments utilizing a pharmacological agent to manipulate PRL in the zebra finch (Taeniopygia guttata) found no support for a role of this hormone in clutch size determination. Here, we take a complementary approach by manipulating clutch size through egg removal, examining co-variation in PRL and LH between two breeding attempts, as well as through experimentally-extended laying. Clutch size increased for egg removal females, but not controls, but this was not correlated with changes in PRL or LH. There were also no differences in PRL between egg removal females and controls, nor did PRL levels during early, mid- or late-laying of supra-normal clutches predict clutch size. By uncoupling PRL, LH and clutch size in our study, several key predictions of the PRL-based mechanistic model for CSD were not supported. However, a positive correlation between PRL levels late in laying and days relative to the last egg (clutch completion) provides an alternative explanation for the equivocal results surrounding the conventional PRL-based physiological model for CSD. We suggest that females coordinate PRL-mediated incubation onset with clutch completion to minimize hatching asynchrony and sibling hierarchy, a behavior that is amplified in females laying larger clutches.

Ovarian follicle development in avian species is characterized by a strict hierarchical arrangement. The hierarchical follicles secrete progesterone, which induces the LH surge, but the capacity to produce other steroids decreases with development. Our aim was to evaluate the complementary action of subdominant follicles (F4-F6) on ovulation and steroidogenesis of the preovulatory follicles (F1-F3) in domestic laying hens. The first study included four groups: control (C); sham-operated (SO); large hierarchical follicles (LHF) from which F4-F6 follicles were extracted; and subdominant hierarchical follicles (SHF) from which F1-F3 follicles were extracted. Blood samples were collected every 2h from 12h before estimated ovoposition until 2h after ovoposition. Egg laying continued at the same rates in C and SO hens, with normal preovulatory surges of oestradiol, testosterone, progesterone and LH. In contrast, in LHF and SHF groups, ovoposition was blocked; oestradiol concentrations were not affected; but no preovulatory surges of testosterone, progesterone or LH were seen. Further, the testosterone surge was required for the occurrence of progesterone and LH surges. In the second study StAR and steroidogenic enzyme mRNA expression was evaluated within F1-F3 follicles from a LHF group and C-14 and C-8 controls groups, in which follicles were collected 14h and 8h before expected ovoposition, respectively. Extraction of F4-F6 follicles caused a significant reduction in StAR and 3β-HSD expressions within theca, but not in granulosa cells. In conclusion, subdominant hierarchical follicles (F4-F6) are required for the preovulatory release of testosterone, progesterone and LH, which are highly inter-correlated.

Clutch size is a fundamental predictor of avian fitness, widely-studied from evolutionary and ecological perspectives, but surprisingly little is known about the physiological mechanisms regulating clutch size variation. The only formal mechanistic hypothesis for avian clutch-size determination predicts an anti-gonadal effect of circulating prolactin (PRL) via the inhibition of luteinizing hormone (LH), and has become widely-accepted despite little experimental support. Here we investigated the relationship between pre-breeding and breeding plasma PRL and LH and clutch-size in captive-breeding female zebra finches (Taeniopygia guttata). Using a repeated-measures design, we followed individual females from pre-breeding, through multiple breeding attempts, and attempted to decrease PRL using the D2-receptor agonist, bromocriptine. Clutch size was independent of variation in pre-breeding PRL or LH, although pre-breeding LH was negatively correlated with the time between pairing and the onset of laying. Clutch size was independent of variation in plasma PRL on all days of egg-laying. Bromocriptine treatment had no effect on plasma PRL, but in this breeding attempt clutch size was also independent of plasma PRL. Finally, we found no evidence for an inverse relationship between plasma PRL and LH levels, as predicted if PRL had inhibitory effects via LH. Thus, our data fail to provide any support for the involvement of circulating PRL in clutch size determination. These findings suggest that alternative models for hormonal control of avian clutch size need to be considered, perhaps involving downstream regulation of plasma PRL at the level of the ovary, or other hormones that have not been considered to date.

Prior experience with photostimulation enhances photo-induced reproductive response in female house finches. The NERC and CEH trademarks and logos ('the Trademarks') are registered trademarks of NERC in the UK and other countries, and may not be used without the prior written consent of the Trademark owner. ABSTRACT 24 In many vertebrates parental age is related to reproductive output with older individuals 25 often performing better (e.g., advanced timing, more offspring) than younger ones. First-26 year birds differ from older birds in that they lack previous experience with the 27 reproductively-stimulatory effects of long day lengths (photostimulation). The goal of this 28 study was to examine whether this age-related increase in reproductive output can be 29 attributed, at least in part, to previous experience with photostimulation in a 30 photoperiodic bird, the female house finch (Carpodacus mexicanus). Specifically, we 31 investigated whether previous experience with photostimulation influences the early 32 stages of reproductive development by quantifying plasma luteinizing hormone (LH), 33 plasma vitellogenin, ovarian follicle size, and immunoreactivity of hypothalamic 34 gonadotropin-releasing hormone (GnRH-I) and vasoactive intestinal polypeptide (VIP). 35 By differentially manipulating photoperiod, we generated two groups of first-year female 36 finches: a photo-experienced group that had been through one photoperiodically-37 induced cycle of gonadal development and regression, and a photo-naïve group 38 exposed to long days since hatch. Both groups were then transferred from long to short 39 days for nine weeks, to ensure full photoperiodic responsiveness, and then 40 photostimulated for four weeks and exposed to conspecific or heterospecific male song 41 starting 90 minutes before sacrifice. Following photostimulation, although photo-42 experienced and photo-naïve groups exhibited similar surges in plasma LH 43 concentrations, circulating vitellogenin levels increased in photo-experienced, but not in 44 photo-naïve birds. After four weeks of photostimulation, egg yolk deposition was 45 observed in two of six photo-experienced birds but in none of the photo-naïve birds. 46 3 After four weeks of photostimulation and exposure to conspecific or heterospecific male 47 song, more GnRH-I-ir cells were seen in the septo-preoptic hypothalamus of photo-48 experienced than of photo-naïve birds. In contrast, there were no differences between 49 the photo-experienced and photo-naïve birds, irrespective of the song type they were 50 exposed to, in numbers of visible VIP-ir cells in the mediobasal hypothalamus. Our 51 results demonstrate that previous photo-experience enhances some of the early stages 52 of photo-induced reproductive development, and that the reproductive neuroendocrine 53 …

In vertebrates, reproductive output often increases with age. Unlike older birds, first-year photoperiodic birds lack experience with the reproductively stimulatory effects of long day lengths (photostimulation). We examined whether age-related differences in annual reproductive development could be partially attributed to previous experience with photostimulation in the photoperiodic house finch (Carpodacus mexicanus). By manipulating photoperiod, we generated 2 groups of first-year females: a photo-experienced group that underwent 1 photoperiodically induced cycle of gonadal development and regression and a photo-naïve group exposed to long days since hatch. We transferred both groups from long to short days and then photostimulated and exposed them to male birdsong prior to sacrifice. Following concurrent photostimulation, both groups exhibited similar plasma luteinizing hormone surges and hypothalamic vasoactive intestinal polypeptide immunoreactivity. In contrast, hypothalamic gonadotropin-releasing hormone immunoreactivity and circulating vitellogenin levels were higher in photo-experienced birds, and yolk deposition occurred in only 2 females, both of which were photo-experienced. Our results demonstrate that photo-experience enhances some aspects of early photo-induced reproductive development and raise the hypothesis that photo-experience may account for at least some age-related variation in reproductive output.

In mammals Orexin-A and -B are neuropeptides involved in the hypothalamic regulation of diverse physiological functions including food intake and the sleep-wake cycle. This generalisation was investigated in meat-(broiler) and layer-type juvenile domestic chickens by immunocytochemical localisation of orexin A/B in the hypothalamus, and by measurements of hypothalamic hypocretin mRNA which encodes for orexin A/B after chronic food restriction, and during the sleep-wake cycle. Orexin immunoreactive fibres were observed throughout the hypothalamus with cell bodies in and around the paraventricular nucleus. No differences were observed in the pattern of immunoreactivity using anti- human orexin-A, or -B antisera. The amount of hypothalamic hypocretin mRNA in food -restricted broilers was higher than in broilers fed ad libitum, but the same as in layer- type hens fed ad libitum. Hypothalamic hypocretin mRNA was increased (P<0.01) in 12-week-old broilers fed 25% of their ad libitum intake between 6–12 weeks of age. No difference in hypothalamic hypocretin mRNA was seen in 12-week-old layer- type hens when they were awake (1–2 h after lights on) or sleeping (1–2 h after lights off). It is concluded that in the chicken, we could not find evidence that hypothalamic orexin plays a role in the sleep-wake cycle and it may be involved in aspects of energy balance.

Many bird species have advanced their seasonal timing in response to global warming, but we still know little about the causal effect of temperature. We carried out experiments in climate-controlled aviaries to investigate how temperature affects luteinizing hormone, prolactin, gonadal development, timing of egg laying and onset of moult in male and female great tits. We used both natural and artificial temperature patterns to identify the temperature characteristics that matter for birds. Our results show that temperature has a direct, causal effect on onset of egg-laying, and in particular, that it is the pattern of increase rather than the absolute temperature that birds use. Surprisingly, the pre-breeding increases in plasma LH, prolactin and in gonadal size are not affected by increasing temperature, nor do they correlate with the onset of laying. This suggests that the decision to start breeding and its regulatory mechanisms are fine-tuned by different factors. We also found similarities between siblings in the timing of both the onset of reproduction and associated changes in plasma LH, prolactin and gonadal development. In conclusion, while temperature affects the timing of egg laying, the neuroendocrine system does not seem to be regulated by moderate temperature changes. This lack of responsiveness may restrain the advance in the timing of breeding in response to climate change. But as there is heritable genetic variation on which natural selection can act, microevolution can take place, and may represent the only way to adapt to a warming world.

The reproductive status of adult Pekin drakes is very sensitive to nutritional status. Thus, the purpose of this study was to increase our understanding of the neurobiology underlying the depressive effect of fasting on the secretion of reproductive hormones. It was hypothesized that this effect was mediated by gonadotropin-inhibitory hormone (GnIH). Networks of GnIH fibers were present throughout the diencephalon, and cell bodies were present primarily, in the hypothalamic paraventricular nucleus (PVN). The duck GnIH gene was cloned and sequenced and found to encode GnIH and two GnIH-related peptides (GnIH-RP1, GnIH-RP2) which have a similar identity to those found in other avian species. Intracerebroventricular injection of GnIH, but not of GnIH-RP1, depressed plasma LH and stimulated feeding. Fasting for 48h depressed plasma LH and induced fos expression in about half the population of GnIH-ir neurons. These data suggest that GnIH neurons are mediators between feeding and reproductive systems in Pekin drakes.

Most animals reproduce seasonally.They time their reproduction in response to environmental cues, like increasing photoperiod and temperature, which are predictive for the time of high food availability.Although individuals of a population use the same cues, they vary in their onset of reproduction, with some animals reproducing consistently early or late.In avian research, timing of reproduction often refers to the laying date of the first egg, which is a key determinant of fitness.Experiments measuring temporal patterns of reproductive hormone concentrations or gonadal size under controlled conditions in response to a cue commonly assume that these proxies are indicative of the timing of egg laying.This assumption often remains untested, with few studies reporting both reproductive development and the onset of laying.We kept in total 144 pairs of great tits (Parus major) in separate climate-controlled aviaries over 4 years to correlate pre-breeding plasma luteinizing hormone (LH), prolactin (PRL) and gonadal growth with the timing of laying.Individuals varied consistently in hormone concentrations over spring, but this was not directly related to the timing of gonadal growth, nor with the laying date of the first egg.The timing of gonadal development in both sexes was similarly not correlated with the timing of laying.This demonstrates the female's ability to adjust the onset of laying to environmental conditions irrespective of substantial differences in pre-laying development.We conclude that stages of reproductive development are regulated by different cues, and therefore egg laying dates need to be studied to measure the influences of environmental cues on timing of seasonal reproduction.

The Challenge Hypothesis postulates that male vertebrates can respond to social challenges, such as simulated territorial intrusions, by rapidly increasing their concentrations of plasma androgens, such as testosterone (T). This increase may facilitate the expression of aggressive behavior and lead to persistence of this behavior even after withdrawal of the challenge, thus potentially promoting territoriality and the probability of winning future challenges. The scope of the Challenge Hypothesis was tested by exposing free-ranging male Cassin's Sparrows, Peucaea cassinii, to conspecific song playback (SPB) at the beginning of the vernal nesting season. Exposure to SPB stimulated aggressive behavior but did not influence plasma T. Furthermore, plasma T did not correlate with the duration of exposure to SPB, and the behavioral response to SPB did not differ in males that were challenged a second time shortly after the first challenge. As birds were investigated at a stage of their reproductive cycle when plasma T is presumably seasonally high due to photostimulation, the lack of hormonal response to SPB may have been due to the hypothalamus-pituitary-gonadal axis secreting hormones at maximum rates. This was not the case, however, because administration of gonadotropin-releasing hormone I rapidly stimulated the secretion of luteinizing hormone (LH) and T, and treatment with ovine LH rapidly stimulated T secretion.

Gonadotropin-releasing hormone (GnRH) is central to the control of vertebrate reproductive cycles and since GnRH orthologs are also present in invertebrates, it is likely that the common ancestor of bilateral animals possessed a GnRH-like peptide. In order to understand the evolutionary and comparative biology of GnRH peptides we cloned the cDNA transcripts of prepro GnRH-like peptides from two species of bivalve molluscs, the Yesso scallop Patinopecten yessoensis and the Pacific oyster Crassostrea gigas. We compared their deduced uncleaved and mature amino acid sequences with those from other invertebrates and vertebrates, and determined their sites of expression and biological activity. The two molluscan GnRH sequences increased the number of known protostome GnRHs to six different forms, indicating the current classification of protostome GnRHs requires further revision. In both molluscs, RT-PCR analysis showed that the genes were highly expressed in nervous tissue with lower levels present in peripheral tissues including the gonads, while immunocytochemistry, using anti-octopus GnRH-like peptide, demonstrated the presence of GnRH-like peptide in neural tissue. Putative scallop GnRH-like peptide stimulated spermatogonial cell division in cultured scallop testis, but the scallop GnRH-like peptide did not stimulate LH release from cultured quail pituitary cells. This is the first report of the cloning of bivalve GnRH-like peptide genes and of molluscan GnRH-like peptides that are biologically active in molluscs, but not in a vertebrate.

Timing of reproduction in temperate-zone birds is strongly correlated with spring temperature, with an earlier onset of breeding in warmer years. Females adjust their timing of egg laying between years to be synchronized with local food sources and thereby optimize reproductive output. However, climate change currently disrupts the link between predictive environmental cues and spring phenology. To investigate direct effects of temperature on the decision to lay and its genetic basis, we used pairs of great tits (Parus major) with known ancestry and exposed them to simulated spring scenarios in climate-controlled aviaries. In each of three years, we exposed birds to different patterns of changing temperature. We varied the timing of a temperature change, the daily temperature amplitude, and the onset and speed of a seasonal temperature rise. We show that females fine-tune their laying in response to a seasonal increase in temperature, whereas mean temperature and daily temperature variation alone do not affect laying dates. Luteinizing hormone concentrations and gonadal growth in early spring were not influenced by temperature or temperature rise, possibly posing a constraint to an advancement of breeding. Similarities between sisters in their laying dates indicate genetic variation in cue sensitivity. These results refine our understanding of how changes in spring climate might affect the mismatch in avian timing and thereby population viability.

Chronic stress generally inhibits the activity of the reproductive system. Acute stress also is often inhibitory, but the mechanism involved and its persistence of action once animals are no longer exposed to the stressor are poorly understood. We investigated the effect of capture and restraint stress on plasma testosterone (T), luteinizing hormone (LH), and corticosterone (CORT) in free-ranging male rufous-winged sparrows, Peucaea carpalis. Stress decreased plasma T between 10 and 30 min after capture and restraint but did not influence plasma LH, the main hormone that controls T secretion, suggesting that stress did not decrease plasma T by inhibiting LH secretion. The stress-induced decrease in plasma T was associated with elevated plasma CORT, but there was no evidence that these effects were functionally related. Plasma stress-induced T was positively related to plasma initial T measured within 2 min of capture. This relationship was, however, complex as plasma T decreased proportionally more in response to stress in sparrows with high than low plasma initial T. The relative sensitivity to the same stressor was, therefore, individually variable and this variation was related to initial plasma T. Birds caught and restrained for 30 min, and then released on their breeding territory before recapture up to 6 h later, maintained depressed plasma T, indicating that the effect of acute stress on this hormone persists after the stressor removal. These studies provide new information on the effects of acute stress on plasma T in free-ranging birds. In particular, they are among the first to characterize the time course and to describe the persistence of these effects. The findings also contribute to identifying factors that are associated with individual differences in plasma hormone levels.

Hormones mediate major physiological and behavioural components of the reproductive phenotype of individuals. To understand basic evolutionary processes in the hormonal regulation of reproductive traits, we need to know whether, and during which reproductive phases, individual variation in hormone concentrations relates to fitness in natural populations. We related circulating concentrations of prolactin and corticosterone to parental behaviour and reproductive success during both the pre-breeding and the chick-rearing stages in both individuals of pairs of free-living house sparrows, Passer domesticus. Prolactin and baseline corticosterone concentrations in pre-breeding females, and prolactin concentrations in pre-breeding males, predicted total number of fledglings. When the strong effect of lay date on total fledgling number was corrected for, only pre-breeding baseline corticosterone, but not prolactin, was negatively correlated with the reproductive success of females. During the breeding season, nestling provisioning rates of both sexes were negatively correlated with stress-induced corticosterone levels. Lastly, individuals of both sexes with low baseline corticosterone before and high baseline corticosterone during breeding raised the most offspring, suggesting that either the plasticity of this trait contributes to reproductive success or that high parental effort leads to increased hormone concentrations. Thus hormone concentrations both before and during breeding, as well as their seasonal dynamics, predict reproductive success, suggesting that individual variation in absolute concentrations and in plasticity is functionally significant, and, if heritable, may be a target of selection.

1. Annual variation in the timing of avian reproduction is associated with predictive cues related to ambient temperature. Understanding how these cues affect timing, and estimating the genetic variation in sensitivity to these cues, is essential to predict the micro-evolutionary changes in timing which are needed to adapt to climate change.2. We carried out a 2-year experiment with great tits Parus major of known genetic background, which were kept in pairs in climate-controlled aviaries with simulated natural photoperiod and exposed to a seasonal change in temperature, where the two treatments differed by 4 degrees C. We recorded the dates of laying the first and last eggs and timing of moult, as well as physiological proxies associated with reproduction: plasma luteinizing hormone (LH), prolactin, and gonadal size at four-weekly intervals.3. The temperature treatments did not affect first-egg dates, nor gonadal growth or plasma LH and prolactin concentrations. However, birds terminated egg laying, regressed their testes and started their moult earlier at higher temperatures.4. There were marked family differences in both the start of egg laying, with sisters from early laying maternal families laying early, and in the termination of laying, indicating that there is heritable variation in sensitivity to cues involved in timing.5. Our experiment, the first to use genetically related individuals in an experimental design with a natural change in photoperiod and biologically realistic temperature differences, thus shows that genetic adaptation in cue sensitivity is possible, essential for species to be able to adapt to a warming world.

In mammals, photoreception is restricted to cones, rods and a subset of retinal ganglion cells. By contrast, non-mammalian vertebrates possess many extraocular photoreceptors but in many cases the role of these photoreceptors and their underlying photopigments is unknown. In birds, deep brain photoreceptors have been shown to sense photic changes in daylength (photoperiod) and mediate seasonal reproduction. Nonetheless, the specific identity of the opsin photopigment ‘sensor’ involved has remained elusive. Previously, we showed that vertebrate ancient (VA) opsin is expressed in avian hypothalamic neurons and forms a photosensitive molecule. However, a direct functional link between VA opsin and the regulation of seasonal biology was absent. Here, we report the in vivo and in vitro absorption spectra ( λ max = ∼490 nm) for chicken VA photopigments. Furthermore, the spectral sensitivity of these photopigments match the peak absorbance of the avian photoperiodic response ( λ max = 492 nm) and permits maximum photon capture within the restricted light environment of the hypothalamus. Such a correspondence argues strongly that VA opsin plays a key role in regulating seasonal reproduction in birds.