Plasticity of brood patch development and its influence on incubation periods in the yellow-eyed penguin Megadyptes antipodes: an experimental approach

In many bird species, eggs laid late in the laying period hatch after a shorter incubation period than early-laid eggs. However, the mechanisms that explain these seasonal declines in incubation periods among clutches remain poorly understood. In this study we investigated the plasticity of brood patch development during incubation in yellow-eyed penguins Megadyptes antipodes and established whether differences exist in brood patch formation among early, mean and late-breeding penguins. We also examined whether brood patch development was influenced by sex and age of birds. We then placed an artificial egg in nests a few days prior to egg laying to investigate whether the presence of an egg influences brood patch development and whether an advanced brood patch development at the time of egg laying causes declines in incubation periods. Initial brood patch width on the day the first egg was laid was dependent on sex and age, while the development of brood patch width after first egg laying was slower in early-laying birds than in mean- and late-laying birds. Initial brood patch temperature as well as temperature throughout incubation was largely dependent upon sex, whereby males had higher brood patch surface temperatures than females. Placement of an artificial egg in nests stimulated successfully brood patch development in manipulated birds, so that by the time they laid their own first egg, their brood patches were wider and had higher temperatures than those of control birds. Moreover, incubation periods of first eggs from manipulated nests were significantly shorter (43.5 days) than were those from control nests (47.3 days). Thus, variation in brood patch development and related differences in incubation temperature during early incubation could contribute to seasonal declines in incubation periods.     

No sex difference in yolk steroid concentrations of avian eggs at laying

Yolk steroids of maternal origin have been proposed to influence genetic sex determination in birds, based on sex differences in yolk steroid concentrations of peafowl eggs incubated for 10 days. More recent reports dispute this proposal, as yolk steroids in eggs incubated for 3 days do not show such sex differences. To date, research examining this phenomenon has only analysed incubated eggs, although sex in avian species is determined before incubation begins. This may be a serious methodological flaw because incubation probably affects yolk steroid concentrations. Therefore, we investigated sex differences in yolk steroid concentrations of unincubated avian eggs. We withdrew yolk for steroid analysis from fresh, unincubated Japanese quail (Coturnix japonica) eggs by biopsy, and then incubated those eggs for 10 days, after which we harvested the embryonic material for genetic sexing and the incubated yolk for further steroid analysis. We found no sex differences in fresh Japanese quail eggs; however, sex differences were apparent in yolk steroids by day 10 of incubation, when female eggs had significantly more oestrogen in relation to androgen than male eggs. Concentrations of all yolk androgens decreased dramatically between laying and day 10 of incubation, whereas oestradiol (E2) concentrations increased marginally. Thus, yolk concentrations of androgens and E2 do not appear critical for avian sex determination.     

Involvement of cholecalciferol metabolism in birds in the adaptation of calcium absorption to the needs during reproduction.

1. The metabolism of calcium and cholecalciferol in quail (Coturnix coturnix japonica) and chicken (Gallus domesticus) during maturation was correlated to gonadal activity and plasma oestrogen levels. 2. Birds with undeveloped ovaries (immature), developed ovaries but not laying (mature), and after laying 3-8 eggs (laying), were used in the first series. 3. Birds in which egg production had been arrested by Nicarbazin, were used in the second series. 4. Plasma 17 beta-oestradiol and calcium were elevated in the mature bird, with no further change in the laying bird. Kidney 25-hydroxycholecalciferol-1-hydroxylase and intestinal calcium-binding protein increased slightly in the mature bird, whereas they were grossly elevated in the laying bird. 5. Calcium and phosphorus absorption were markedly elevated in the laying bird. 6. No changes were noted in plasma 25-hydroxycholecalciferol, at any stage of maturation. 7. During the arrest of egg production by Nicarbazin, 17 beta-oestradiol level, calcium concentration of plasma, and medullary bone were maintained. Kidney 25-hydroxy-cholecalciferol-1-hydroxylase, intestinal calcium-binding protein and absorption of calcium were strikingly reduced. 8. The results suggest that changes in calcium absorption and cholecalciferol metabolism during maturation in birds are not directly affected by gonadal hormones; they appear to represent an adaptation to the increased calcium needs due to medullary bone formation and, more importantly, to the large losses of calcium imposed by shell formation.     

Egg temperature and initial brood patch area determine hatching asynchrony in Magellanic penguin Spheniscus magellanicus

In birds, the adaptive significance of hatching asynchrony has been under debate for many years and the parental effects on hatching asynchrony have been largely assumed but not often tested. Some authors suggest that hatching asynchrony depends on the incubation onset and many factors have been shown to influence hatching asynchrony in different species. Our objective was to analyze the exact timing of the onset of incubation and if this affects hatching asynchrony; and, in addition, which other factors (brood patch development, incubation position, adult body condition, intra‐clutch egg dimorphism, laying date and year) affect hatching asynchrony in Magellanic penguins Spheniscus magellanicus. We first estimated the eggshell temperature at which embryo development starts, with a non‐destructive and novel method. We then recorded individual egg temperatures in 61 nests during incubation, and related them, and other breeding parameters, to hatching asynchrony. We also observed incubation positions in 307 nests. We found a significant positive relationship between hatching asynchrony and the temperature that the first‐laid egg experienced during egg laying and between hatching asynchrony and the initial brood patch area. We also found a negative relationship between hatching asynchrony and the difference in temperature between second and first‐laid eggs within a clutch, measured after the egg‐laying period was finished. We ruled out position of the eggs during incubation, adult body condition, egg volume, laying date, and study year as factors influencing hatching asynchrony. The egg temperature during laying and the difference in temperature between eggs of a clutch are determinants of hatching asynchrony in Magellanic penguins.     

Effect of artificially increased day length in winter on female domesticated canaries

Three groups of female canaries were given different schedules of increasing day-length between October and December: all groups responded in the same way. Three groups of female canaries were given a 20 hour light/four hour dark schedule at different periods outof the breeding season (October-December, October-January, and January-February).All experimental birds became defeathered and vascular. There was an increase in skin tactile sensitivity in October-December but not in January-February. Nest-building was induced in all months. Only six out of 41 experimental birds laid eggs. Eleven birds which did not lay showed marked incubation behaviour. Discussion centres around the hormonal control of brood patch formation. The possibility that the absence of an effect of photostimulation on sensitivity in January-February is due to shortage of progesterone is considered. These light schedules induced more nest-building than injected oestrogen: no conclusions are yet possible on the hormonal control of next-building.     

Culture of naked quail (Coturnix coturnix japonica) ova in vitro for avian transgenesis: Culture from the single-cell stage to hatching with pH-adjusted chicken thick albumen

We first examined the pH change of the albumen of quail (Coturnix coturnix japonica) eggs before and after they were laid, as well as that of laid eggs. The pH rose rapidly after laying and continued to increase gradually in storage. Incubation at 37.5°C accelerated the increase in the pH of infertile eggs, while that of fertile eggs remained low during incubation. Referring to these results, we obtained a protocol for producing quail hatchlings by culture in vitro from naked ova. The naked ovum was filled with chicken (Gallus domesticus) thick albumen, the pH of which had been adjusted to 7.2–7.4. The ovum was cultured at 41.5°C in 20% CO₂ in air for the first 24 h. Then the embryo was moved to a surrogate quail egg shell that had been filled with non-pH-adjusted chicken thin albumen and cultured for a further 48 h in 100% air. The embryo was transferred again to a surrogate chicken egg shell and cultured under the same conditions until hatching. The culture yielded quail chicks with a hatchability of 19.4%. The method proposed here should be applicable to the production of transgenic birds.     

Is the additional effort of renesting linked to a hormonal change in the common tern?

The production of and care for a replacement clutch can bear costs in terms of future reproduction or survival. However, renesting is quite common among seabirds and can contribute considerably to individual fitness. Prolactin and corticosterone are two hormones involved in the mediation of breeding behavior and, as they are linked to body condition or effort, it is of interest if these hormone values change during a second demanding breeding phase within a year. We compared baseline prolactin and corticosterone between the first and the renesting attempt in common terns (Sterna hirundo) on individual level. Therefore, in addition to control birds, 37 breeders were sampled during incubation of their first and their replacement clutch in 2008 and 2009. Blood samples were taken non-invasively by blood-sucking bugs. Prolactin level was lower during the renesting period, especially in birds which abandoned their clutch afterwards, whereas corticosterone did not change. Excluding the deserting birds, the reduced prolactin level was not linked to minor success, but could be related to seasonal processes. The control group of late laying common terns showed comparably low prolactin values, but increased corticosterone concentrations. Renesting individuals exhibited higher prolactin during incubation of their first clutch than non-renesting birds, probably indicating their higher quality. The fact that terns still have relatively high prolactin and low corticosterone values during renesting might confirm their higher quality and suggests that they are able to meet the costs of a second demanding breeding period without being considerably stressed.     

Regulation of prolactin and its role in gallinaceous bird reproduction

There are major changes in circulating luteinizing hormone (LH), prolactin (PRL), estrogens (E), and progesterone (P) in relation to the onset of reproduction, egg laying, incubation, and care of young. LH levels increase in the prelaying period, followed some days later by increased circulating levels of E, P, and PRL. Levels of these hormones tend to stabilize during egg laying with periodic ovulatory cycle changes. Around the onset of incubation PRL levels increase, while LH, E, and P levels fall. During incubation PRL reaches very high levels, falling sharply when incubation is terminated. Stimulatory effects of hypothalamic neurotransmitters, peptides, and ovarian steroids on PRL secretion have been shown. The prelaying increase is dependent on E and P and the high levels of incubation require a functional serotonergic system. The causal relationships and roles of PRL in incubation of gallinaceous birds are, however, still unclear.     

Endocrine correlates of parental care in an Antarctic winter breeding seabird, the emperor penguin, Aptenodytes forsteri

Plasma levels of luteinizing hormone (LH) and prolactin associated with parental behavior were measured in the Antarctic winter breeding emperor penguin, Aptenodytes forsteri. Males exclusively incubate the egg while females exclusively brood the nonhomeothermic young; both sexes alternate in rearing the homeothermic young. Birds were sampled on arrival from the sea through egg laying, incubation, and brooding. All parent birds lost their chicks at the end of the brooding period due to harsh weather but sampling continued. In females, LH titers dropped after egg laying but levels were restored when the birds returned from the sea to brood the chicks and were not depressed by high prolactin levels. Plasma prolactin remained low in males captured on arrival and kept until the free-living males finished incubation. In breeders, prolactin secretion increased during the prelaying period when day length decreased. Prolactin levels stayed elevated in males during incubation and in brooding females returning after a 2-month absence at sea. Prolactin values were higher in brooding females than in males ending incubation or returning in late brooding. These levels did not drop after chick loss, and the sexual difference in prolactin values was maintained after breeding failure. In emperor penguins, increased prolactin secretion appears to be triggered around the time of egg laying and continues, driven by an endogenous mechanism, through incubation and brooding until rearing is completed. Prolactin secretion independent of external stimuli may have evolved in pelagic seabirds to maintain parental care despite long absences at sea from the breeding colony.     

Intraclutch egg-size variation in the Barnacle Goose Branta leucopsis: an egg-removal experiment

We carried out an egg-removal experiment on Barnacle Geese Branta leucopsis to test the hypothesis that tactile contact between the brood patch and eggs accumulating in the nest during laying was causally related to intraclutch egg-size variation, acting through the development of incubation behaviour. Egg removal had no effect on mean egg mass, relaying interval or clutch size: mean (±s.e.) clutch sizes for first clutches were 4.9 ± 0.9 and 4.7 ± 0.9 eggs for control and experimental females, respectively. We therefore conclude that Barnacle Geese have a determinate pattern of egg laying. Daily removal of all eggs from the first-laid egg did not affect the within-clutch pattern of egg-size variation: in both control and experimental clutches the first egg was relatively small, the second or third egg was largest and there was a linear decline in egg size to the smallest, last-laid egg. There was no significant difference in either the absolute or relative size of the last-laid eggs in control and experimental clutches. Tactile contact with eggs in the nest, therefore, is not required for the expression of intraclutch egg-size variation in Barnacle Geese. In this respect, determinate-laying Barnacle Geese differ from species with indeterminate laying patterns (e.g. gulls) where egg removal does affect the normal pattern of within-clutch egg-size variation. If hormonal changes (e.g. prolactin) associated with onset of incubation are causally related to the intraclutch decrease in egg size, then the stimuli involved appear to differ between determinate and indeterminate layers.     

Concentrations of plasma prolactin and luteinizing hormone following nest deprivation and renesting in ring doves (Streptopelia risoria)

Ring doves with increased plasma prolactin and low plasma LH (Group A) or with low plasma prolactin and low plasma LH (Group B) which had been incubating sterile eggs for 12 or 18 days, respectively, had their nests and eggs removed for 3 days. Upon nest return, observations were made on the birds' readiness to renest and on changes in plasma prolactin and LH. Birds from Group A demonstrated a far greater tendency to resume incubation than birds from Group B. Nest deprivation resulted in a sharp fall in the concentration of plasma prolactin in birds which were deprived of their nests after 12 days of incubation (Group A). Following resumption of incubation no subsequent increase in the prolactin levels was observed in Group A or B. The concentration of plasma LH rose sharply after nest deprivation in both sexes of both groups and declined after return of the nests. Birds in Groups A and B which returned to their nests laid a new clutch of eggs while continuing to incubate. The total length of uninterrupted sitting following nest return was 20.9 +/- 0.48 days (n = 8). These results suggest that (1) once the mechanism responsible for the increase in plasma prolactin during incubation is disrupted, it cannot be reactivated unless the whole reproductive cycle is repeated. (2) The inhibition of LH secretion during incubation involves neural mechanisms which do not necessarily involve the anti-gonadotrophic action of prolactin.     

Thyroxine can mimic photoperiodically induced gonadal growth in Japanese quail

Summary Pharmacological doses of thyroxine are able to mimic the effects of long photoperiods in Japanese quail. In birds maintained on short daylengths thrice-weekly injections of 100 g thyroxine cause full testicular maturation at rates not greatly different from those seen if quail are exposed to long days. Thyroxine stimulates increases in the secretion of FSH and LH, in pituitary prolactin content and in the hypothalamic content of Gn-RH. The effects are dose-dependent. If female quail kept on short daylengths are given thyroxine their ovaries develop and they lay eggs. In castrated male quail on short days thyroxine causes a ten-fold increase in circulating LH within a week. Thyroxine injections are also capable of maintaining quail in a photorefractory state even when they are transferred to short daylengths. The results suggest that thyroxine mimics long days by acting high in the photoneuroendocrine system and does not simply act to facilitate hormone secretion per se. This is in line with growing evidence in mammals and birds that parts of the photoperiodic machinery are sensitive to thyroid hormones.Abbreviations Gn-RH gonadotropin releasing hormone - FSH follicle stimulating hormone - LH luteinizing hormone - T ₄ thyroxine     

The role of partial incubation and egg repositioning within the clutch in hatching asynchrony and subsequent effects on breeding success

The main mechanism to achieve hatching asynchrony (HA) for incubating birds is to start heating the eggs before clutch completion. This might be achieved through partial incubation and/or early incubation. Even in the absence of incubation behaviour during the laying phase, clutches still experience a certain degree of asynchrony. Recent studies have shown that eggs located in the centre of the nest receive more heat than peripheral ones during incubation. As eggs receiving more heat would develop faster, we hypothesized that HA should be shorter in nests where eggs were moved homogeneously along the centre–periphery space during incubation than in those nests where eggs repeatedly remained in the same locations, either centrally or peripherally. We explored the relative roles of egg repositioning and partial incubation in determining HA in wild birds by (1) removing eggs from 20 Great Tit Parus major nests on the day of laying and replacing them with fake eggs to avoid partial incubation, and returning them when full incubation began; (2) monitoring twice a day the position of each individually marked egg relative to the clutch centre during incubation, and estimating the coefficient of variation of the distances; and (3) determining HA in each nest. Preventing partial incubation reduced HA by 51% days in experimental nests. It also caused negative effects for the incubating females (lengthening the full incubation period) and positive effects for the brood (increasing fledging success). However, our hypothesis about the role of egg repositioning on HA was not supported: all the females moved the eggs with remarkable consistency, generally attaining a coefficient of variation of the distances around 33%, and it was not related to the HA experienced. We therefore conclude that partial incubation is an important factor regulating HA, and females compensate for the potential effects of differential heating by moving the eggs homogeneously within the clutch.     

Hypothalamic monoamine turnover in ring doves (streptopelia risoria), courting,incubating and brooding young

1. The turnovers of hypothalamic 5-hydroxytryptamine (5HT), dopamine (DA) and noradrenaline (NE) were measured in male and female ring doves (Streptopelia risoria) at three stages of the breeding cycle: courtship, 3 days after pairing; early incubation, 1–2 days after egg laying; and brooding, 1–3 days after the squabs had hatched.2. In both sexes plasma LH decreased progressively from courtship through incubation to brooding young. Crop sacs were fully developed in doves brooding young but not at other stages of the reproductive cycle, indicating increased concentrations of plasma prolactin.3. The turnovers of 5HT and DA in both sexes were significantly higher (P<0.001) in doves brooding young than in birds incubating eggs or nest building. The turnover of DA was higher in females than in males at the onset of incubation. The turnover of NE was lower (P > 0.01) in females at the onset of incubation than during courtship or brooding.4. Increased turnover of hypothalamic DA may be more closely related to brooding behaviour than to changes in prolactin or LH secretion.5. Increased hypothalamic 5HT turnover in brooding doves appears to be more directly related to crop sac development, and by inference increased prolactin secretion, than to depressed plasma LH concentrations.     

Seasonal declines in incubation periods of Brünnich's Guillemots Una lomvia:testing proximate causes

In many species of bird, eggs laid late in the laying period hatch after a shorter incubation period than do eggs laid early. These seasonal declines in incubation period are generally thought to confer evolutionary advantages, but the proximate mechanisms that underlie them are poorly understood. Seasonal declines in incubation period are usually attributed to: (1) seasonal increases in ambient air temperatures; (2) seasonal changes in the behaviour of incubating birds; and/or (3) seasonal declines in egg size. In a previous study, Common Guillemot Una aalge incubation periods declined with laying date at a low-Arctic colony. As there was no support for hypotheses 1 or 2, it was suggested that this occurred because egg size declined with laying date, but eggs were not measured in that study. We recorded similar seasonal declines in the incubation periods of the single eggs laid by Brünnich's Guillemots Una lomvia at two low-Arctic colonies in four years. Neither seasonal variation in ambient air temperatures, nor in the behaviour of incubating adults, appeared to cause the declines. As predicted for Common Guillemots, incubation period increased with egg size among Briinnich's Guillemots, in one of two years. However, incubation period declined with laying date in the absence of corresponding declines in egg size. We conclude that none of the three commonly proposed proximate mechanisms adequately explains the seasonal variation in guillemot incubation periods. Several testable, alternative mechanisms are explored.     

Thyroid Hormone Deposition in Avian Eggs and Effects on Embryonic Development

Studies to date indicate that thyroid hormones are present inthe eggs of chickens and quail and that those hormones are primarilyin the yolk. Quail hens deposit thyroid hormones into eggs inproportion to their own thyroid status, but appear to show someregulation of this process. Indirect studies suggest that thyroidhormones are transferred into oocytes bound to lipoproteinsand transthyretin, both of which are taken up by receptor-mediatedprocesses. Thyroid hormones bound to yolk lipoproteins may enterembryos with yolk that is taken up by non-specific endocytosisor they may be transported into the embryo by specific carriers.To date most of these ideas about thyroid hormone transportinto egg yolk and from egg yolk into embryos have not been investigateddirectly. In quail, very high T₄ content of eggs is associatedwith accelerated differentiation and growth of embryonic pelviccartilage, a thyroid hormone-responsive tissue. We evaluatethese effects on embryonic tissues and the changes in yolk hormonecontent during incubation in relation to the timing of thyroiddevelopment and studies of the capability for tissue responsesto thyroid hormones during early embryonic life     

Genetic differences in yolk testosterone levels influence maternal hormone deposition in the second laying cycle in Japanese quails

Maternally-derived yolk androgens exhibit distinct among- and within-female variations but limited data refer to inter-seasonal changes of maternal hormones in the yolk. We investigated the deposition of yolk testosterone (T) across two laying cycles in Japanese quail. To test how genetically-determined differences influence between cycle variations in yolk androgens we compared females from low (LET) and high (HET) egg T lines at the end of the first and at the beginning of the second laying cycle after an induced moult. Line differences in yolk T levels exhibited high consistency exceeding two reproductive cycles. Yolk T concentrations increased in the second laying cycle in HET but not in LET females. Plasma T levels did not differ between cycles in both lines and no line differences were found either before or after the moult indicating the presence of mechanisms limiting the increase of T concentrations in the circulation. Differences in the yolk T levels were not accompanied by changes in the egg and yolk mass. The HET quail laid eggs with heavier eggshell than the LET quail. Our results demonstrate different abilities of mothers to deposit T in their eggs over two reproductive seasons with expected consequences on the development of their progeny.     

A control taste aversion experiment on predators of roseate tern (Sterna dougallii) eggs

European starlings (Sturnus vulgaris) are responsible for high rates of egg predation at one of the main colonies of the endangered roseate tern (Sterna dougallii) in the Azores archipelago. Control taste aversion has been effective in controlling raven predation in a colony of California least tern (Sterna antillarum browni), but there is little quantitative information about its efficacy on other species of predators taking eggs. We conducted a control taste aversion experiment on yellow-legged gulls (Larus michahellis) and European starlings eating eggs of terns in a mixed colony of common (Sterna hirundo) and roseate terns in the Azores. We treated quail (Coturnix coturnix) eggs with methiocarb and deployed them in artificial nests in the tern colony. On the first experiment, conducted before the terns laid eggs, predation rates on quail eggs by yellow-legged gulls showed significant and rapid decrease after deployment of treated eggs. During the second experiment, after the terns had started laying, results were mixed. Although predation rates by European starlings on treated quail eggs decreased, predation rates on tern eggs did not. We conclude that control taste aversion using methiocarb-treated eggs is likely to reduce depredation by gulls but not starlings because of the need to pre-train the birds and the tendency of starlings to be attracted by the movement of adults, not the presence of nests.     

Serum levels of luteinizing hormone, prolactin, estradiol and progesterone in laying and nonlaying mallards (Anas platyrhynchos)

Temporal changes of circulating serum hormones were measured to compare the reproductive endocrinology of laying and nonlaying mallards. In this study all sixteen control mallards left with their mates laid eggs, while only one of sixteen mallards stressed by daily movement into new pens, laid eggs. Serum levels of luteinizing hormone (LH), prolactin, estradiol, and progesterone were significantly lower (P less than 0.05) in stressed nonlaying mallards than in laying mallards over the 7-week period. Within 1 week of the rotation treatment, LH concentrations in stressed mallards averaged (means +/- SEM) 2.72 +/- 0.19 ng/ml and were significantly lower (P less than 0.05) than LH levels in the controls (3.62 +/- 0.18 ng/ml). After 7 weeks, injections of luteinizing hormone releasing hormone (LHRH) induced a greater change in circulating LH levels in stressed mallards (2.1 +/- 0.3 ng/ml) than in breeding control mallards (0.9 +/- 0.2 ng/ml). These data demonstrate that the lack of reproduction in stressed mallards was associated with LHRH-sensitive pituitary pools of LH, despite their low concentrations of serum LH. These data suggest that the block in reproduction is a failure of the hypothalamus to produce or release releasing hormones. The serum hormone levels of the control mallards varied temporally with stages in the nesting cycle. LH levels increased with the onset of nesting activity, and showed marked fluctuations during the laying period. LH levels fell at the onset of incubation but increased after loss of clutch. Estradiol levels were highest prior to the laying of the first egg and their peak coincided with the initial nest building behavior of the females. Progesterone levels increased sharply with the laying of the 2nd-4th eggs, decreased sharply with the laying of the 6th egg, and then increased slightly at the end of the nesting cycle. Prolactin levels were initially low but gradually increased with laying and incubation activity, declined with loss of clutch, and increased again with renesting activity. Prolactin levels in the stressed mallards also increased (P less than 0.01) over the 7-week period, but significantly less (P less than 0.05) than in layers.