K. A. WILLIAMSON,* P. F. SURAI and J. A. GRAVES*

Functional Ecology 2006 Yolk antioxidants and mate attractiveness in the Zebra Blackwell Publishing Ltd Finch K. A. WILLIAMSON,* P. F. SURAI and J. A. GRAVES* *School of Biological Sciences, University of St Andrews, St Andrews KY16 9TS, Scotland, UK and Avian Science Research Centre, Scottish Agricultural College, Auchincruive, Ayr KA6 5HW, Scotland, UK Summary 1. Egg laying is costly for birds, as eggs must contain all the energy and nutrients needed by the developing embryo. As nutrients are limiting, there is a conflict between provisioning eggs and conserving nutrients for future health and survival. 2. Life-history theory suggests that females should choose how to invest resources in relation to the value of the reproductive attempt. Females may manipulate egg composition in relation to the attractiveness of their mate, ensuring chick survival and assuming that the offspring will themselves be more attractive and have higher reproductive success. Antioxidants are an important component of egg composition: they are vital for embryonic and posthatching chick development and survival. 3. Here we demonstrate that female Zebra Finches (Taeniopygia guttata) mated to attractive males deposit more carotenoids and alpha-tocopherol (vitamin E) in later-laid eggs, shown by a significant interaction between laying order and antioxidant concentration. This bias may increase the probability of chick survival from later-laid eggs and potentially the lifetime reproductive success of the laying female. 4. The body condition of the laying female, measured as fat reserves, was found to be a significant predictor of yolk carotenoid concentration but not of yolk alpha-tocopherol concentration. Key-words: alpha-tocopherol, carotenoids, differential allocation, maternal effects Functional Ecology (2006) doi 10.1111/j.1365-2435.2006.01087.x Ecological Society Introduction Females choosing males on the basis of attractiveness have been assumed to gain good genes that may improve offspring viability and fitness (Andersson 1992; Sheldon 2000). However, it has been established that females can influence offspring fitness themselves through their behaviour or the resources they provide. These maternal effects are known to be widespread (Mousseau & Fox 1998), and it is particularly interesting that females may selectively direct them towards offspring that have a higher reproductive value. Therefore the offspring of attractive males may be fitter due to the genetic inheritance from their father, but also due to maternal effects. Burley termed this increased investment differential maternal allocation (Burley 1988). Eggs consist largely of protein, lipids and water, but also contain a number of micronutrients including immunoglobulins, lysozyme, hormones and antioxidants, present at comparatively low concentrations. Birds must acquire these nutrients from the diet or Author to whom correspondence should be addressed. E-mail: kaw8@st-and.ac.uk from body reserves. As depletion of body reserves for egg formation can have considerable consequences for the condition and survival of the laying female (Nager, Monaghan & Houston 2001; Veasey, Houston & Metcalf 2001), there will be a trade-off between investment in eggs and in body maintenance. A female with an attractive mate may allocate more resources to the current breeding attempt, ensuring that her offspring have the greatest possible chance of survival and reproduction, but at an increased cost to her own future survival and/or reproductive success (Burley 1988; Sheldon 2000). In support of this, females have been found to lay larger clutches (Petrie & Williams 1993) or bigger eggs (Cunningham & Russell 2000) when mated to a preferred male. Differential investment in egg laying has also been found to involve the selective allocation of micronutrients such as androgens and immunoglobulins (Gil et al. 1999; Saino et al. 2002a). Antioxidants are micronutrients that constitute a component of egg quality and offer protection against the damaging effects of free radicals (Halliwell & Gutteridge 1999; Surai 2002). Free radicals are produced as part of normal body processes, the most important contributor 354

355 Yolk antioxidants and mate attractiveness being cellular respiration. They are unstable, extremely reactive and cause damage to DNA, proteins, lipids and carbohydrates that result in tissue injury, disease and cell death. Antioxidants form a defence against the damaging effects of free radicals (Halliwell & Gutteridge 1999; Surai 2002). Two groups of antioxidants are found within eggs: water-soluble antioxidants found in the albumen; and lipid-soluble antioxidants in the yolk. Lipid-soluble antioxidants include vitamin E and carotenoids, both of which are synthesized only by plants; animals must obtain them from their diet. This suggests that they may be limiting, and a source of potential conflict between maintaining body reserves and the demands of egg laying. In a study on Lesser Black-backed Gulls (Larus fuscus), Blount et al. (2004) showed that dietary carotenoid supplementation enhanced egg-laying capacity. Carotenoids are a group of over 600 biologically active pigments that produce colours ranging from dark red to pale yellow, and are responsible for the colour of egg yolk (Surai 2002). Vitamin E is the generic term given to a group of substances with similar biological structure and chemical properties. Within this group, alpha-tocopherol has the greatest antioxidant potential (Halliwell & Gutteridge 1999; Surai 2002). Vitamin E and carotenoids play an important role in embryonic growth, hatching success and the development and survival of the chick (Surai 2002). Carotenoids are important for cell differentiation and proliferation, and function best at low oxygen tensions such as those found in the developing embryo. Eggs with low levels of vitamin E show reduced fertility, slowed embryonic growth and reduced viability of newly hatched chicks (Surai 2002). Egg yolk contains high levels of lipids that provide most of the energy requirements of the embryo. These lipids are also constituents of biological membranes and precursors of substances such as hormones (Surai 2002). Free radicals can destroy important yolk lipids through peroxidation. Antioxidants prevent this, allowing lipids to be used during embryonic growth, or stored for use after hatching for the development of the immune, endocrine and digestive systems during the first week of life. Both carotenoids and vitamin E are also immunostimulants. On hatching, the main immune defence is that of maternally derived immunoglobulins. Antioxidants protect this passively acquired immunity, and chicks hatching from eggs enriched with vitamin E and carotenoids have an increased immune response (Haq, Bailey & Chinnah 1996; Surai 2002). Given the importance of antioxidants both for chick development and in maintaining the health of the laying female, we predicted that female Zebra Finches would deposit higher amounts of alpha-tocopherol and carotenoids in egg yolk when mated to attractive males than when mated to unattractive males. Attractiveness in Zebra Finches can be manipulated using coloured leg rings. Females prefer males wearing red leg rings and avoid those wearing green leg rings (Burley, Krantzberg & Radman 1982). To examine potential differences in antioxidant allocation with mate attractiveness, males from our stock population were given either red or green leg rings, making them either attractive or unattractive. These males were then paired randomly with females and allowed to produce a clutch of eggs. Eggs were collected on the day of laying and the level of alpha-tocopherol and carotenoids in yolk was measured using high-performance liquid chromatography (HPLC). To date, a limited number of studies have examined yolk antioxidant levels in non-domesticated bird species. These studies show that concentrations of alphatocopherol and carotenoids decrease with laying order (Royle, Surai & Hartley 2001, 2003). Two studies have explored the relationship between social environment and yolk antioxidant levels. Verboven et al. (2005) found that female Lesser Black-backed Gulls exposed to high frequencies of aggressive interactions deposited higher levels of carotenoids in eggs containing female embryos compared with eggs laid by controls. Saino et al. (2002b) examined the effects of mate attractiveness on yolk carotenoid concentration in the Barn Swallow (Hirundo rustica). Females mated to unattractive males laid eggs with higher carotenoid concentrations in first-laid eggs than those mated to attractive or control males. However, this study examined only first- and fourthlaid eggs, and the concentration of a single carotenoid, lutein, was measured. We examined the concentration of carotenoids and alpha-tocopherol in all eggs within the laying sequence. We measured total carotenoid levels and did not distinguish between individual carotenoids. Methods GENERAL METHODS The female Zebra Finches used in this experiment were bred at St Andrews University and were of known breeding history. Females were of similar age, and where possible we used females with no previous breeding experience (eight out of ten females) to eliminate potential carry-over effects of mate quality or mate attractiveness from an earlier breeding attempt (Rutstein et al. 2004). The remaining two females had bred only once, at least 6 months previously, with males whose attractiveness had not been manipulated. Females were weighed, tarsus measurements were taken and they were given a fat score. Fat scores give an indication of body condition, and are determined by visually scoring the amount of fat stored in the furculum between the clavicles. Scores ranged from 0 to 6, with 0 = no fat; 6 = convex and overflowing the length of the furculum (Helms & Drury 1960). Female Zebra Finches were paired with males whose attractiveness had been manipulated using coloured leg rings (Burley et al. 1982). Ten males were

356 K. A. Williamson et al. taken at random from our stock population; five were allocated red leg rings and five green leg rings (A.C. Hughes, Middlesex, UK). Previous choice tests on females in our stock population established that they prefer red-ringed over green-ringed males (unpublished data). Pairs were housed in individual breeding cages measuring 75 40 40 cm, with an open nest box and nesting material. This prevents assortative mating where the best quality females may choose the most attractive males. Rooms were maintained at 21 C under full spectrum lighting with a photoperiod of 14 h light : 10 h dark. Water and mixed seed (Haith s Foreign Finch Mix, Cleethorpes, UK) were available ad libitum. Twice weekly supplements of Haith s egg biscuit and fresh spinach leaves were provided. All birds were maintained on the same diet both before and during breeding. Nest boxes were checked twice daily. Eggs were removed on laying and replaced with dummy eggs. They were then weighed, measured, numbered using a non-toxic pen and stored at 70 C awaiting yolk analysis. YOLK ANALYSIS Yolk antioxidant concentrations were determined using HPLC. Eggs were dissected from frozen and yolks were weighed on a digital balance to the nearest 0 001 g. A sample of yolk (100 mg) was mixed with 0 7 ml 5% NaCl solution and 1 ml ethanol, 2 ml hexane was added and the mixture was homogenized for 30 s. Samples were then centrifuged and the hexane phase containing vitamin E and carotenoids was collected. Extraction with hexane was repeated and the combined collection was dried under N 2 gas. The resulting residue was dissolved in methanol/dichloromethane (1 : 1 v/v) and centrifuged ready for HPLC analysis. Carotenoid concentrations were determined using a Spherisorb type S30DS2, 5-µm C18 reversephase column, 25 cm 4 6 mm (Phase Separation, Clwyd, UK) with a mobile phase of methanol/water (97 : 3 v/v) at a flow rate of 1 50 ml min 1. Total carotenoids were detected as a single peak at 445 nm. For vitamin E, chromatography was performed using a Spherisorb type S3ODS2 3-µm C18 reverse-phase column and mobile phase of methanol/water (97 : 3 v/v) at a flow rate of 1 05 ml min 1. Fluorescence detection of vitamin E involved excitation and emission wavelengths of 295 and 330 nm. Peaks were identified by comparison with standards. mate attractiveness and laying order. Mixed linear models allow the inclusion of a random factor: in this case female identity. This takes into account that all eggs within a clutch originate from the same female and are subsequently related. They are therefore nested within each female and are not treated as independent measures. Mate attractiveness, laying order, female fat score and tarsus length were included as the main effects. By including female fat score and tarsus length in the model, it was possible to control statistically for any confounding effects of female body size and condition. Following a forward stepwise procedure, tarsus length was eliminated statistically from all models at an early stage. Data were tested for normality using Anderson Darling tests and transformed where appropriate. Analysis used SAS v8e (1998) (SAS Institute, Cary, NC, USA). Final models were checked for goodness of fit using residual plots (Grafen & Hails 2002). Results We found no change with laying order in either egg weight (F 1,37 = 0 05, P = 0 82) or proportion of yolk (F 1,37 = 0 32, P = 0 58). Female body condition, measured as fat score, had a significant effect on the concentration of carotenoids within eggs. Yolk carotenoid concentration increased with female fat score (F 1,36 = 7 41, P = 0 0099; Fig. 1). Carotenoid concentration decreased with laying order (F 1,36 = 166 88, P 0 0001). Analysis of the overall quantity of carotenoids for all eggs in a clutch together showed that investment in the clutch as a whole did not differ between those laid for attractive and unattractive males (F 1,6 = 4 95, P = 0 068). However, a significant interaction between mate attractiveness and laying order showed that the pattern of carotenoid deposition across laying order differed with mate attractiveness. Females mated to attractive males deposited fewer carotenoids in earlier-laid eggs and more in the last-laid egg than females mated to unattractive males (F 1,36 = 6 79, P = 0 013; Fig. 2). The total amount of carotenoids in whole yolk also showed a significant interaction between mate attractiveness and laying order (F 1,36 = 4 08, P = 0 05). STATISTICAL ANALYSIS The effect of mate attractiveness on total carotenoid and alpha-tocopherol levels for all eggs within each clutch as a whole were compared using general linear models. Clutch size, female fat score and mate attractiveness were included as the main effects. Mixed general linear models were used to assess any differences in the amount of yolk antioxidants with Fig. 1. Mean yolk carotenoid concentration fitted from the statistical model (µg g 1 ± SE) with female fat score.

357 Yolk antioxidants and mate attractiveness Fig. 2. Mean yolk carotenoid concentration fitted from the statistical model (µg g 1 ± SE) in eggs laid for attractive and unattractive males. Fig. 3. Mean yolk alpha-tocopherol concentration fitted from the statistical model (µg g 1 ± SE) in eggs laid for attractive and unattractive males. Alpha-tocopherol concentration decreased significantly with laying order (F 1,36 = 57 71, P 0 0001). However, female condition, measured as fat score, had no effect on the concentration of alpha-tocopherol in eggs (F 1,37 = 0 53, P = 0 47). Overall investment of alpha-tocopherol in the clutch as a whole showed no difference between clutches laid for attractive or unattractive males (F 1,7 = 0 67, P = 0 44). There was a significant interaction between mate attractiveness and laying order, indicating that females mated to attractive males deposited less alpha-tocopherol in first-laid eggs than females mated to unattractive males, but more in later-laid eggs (F 1,36 = 5 41, P = 0 026; Fig. 3). For total amount of alpha-tocopherol in whole yolk, there was also a significant interaction between mate attractiveness and laying order, showing the same pattern of allocation across the clutch (F 1,36 = 4 36, P = 0 044). Discussion Life-history theory predicts that females should allocate resources to reproduction in a manner that increases their lifetime reproductive success (Stearns 1992; Lessells 1991). Females are expected to make a greater investment when mated to an attractive male, provided this investment will increase the success of their offspring (Burley 1988). Antioxidants are an important resource: they are required by the laying female to maintain her health, but they are also needed to ensure successful embryonic and posthatching development and survival of the offspring. The lipidsoluble yolk antioxidants carotenoids and vitamin E are gained from the diet, suggesting that they are limiting and therefore a source of a potential conflict for the female between investment in her eggs and body maintenance. In agreement with other studies, yolk carotenoid and alpha-tocopherol concentration decreased with laying order (Royle et al. 2001, 2003). Dietary and supplemental feeding studies show that females on an enhanced diet deposit higher levels of carotenoids and alpha-tocopherol in yolk, which would be expected as these antioxidants are derived from the diet (Blount et al. 2002; Royle et al. 2003). However, the pattern of decrease with laying order is not affected, suggesting that it is a constraint of the egg laying process. We did not find that females mated to attractive males deposited more antioxidants in the clutch as a whole, which may suggest that females are limited in the overall amount of antioxidants that can be made available for clutch formation. However, the significant interaction between mate attractiveness and laying order showed that the pattern of both carotenoid and alphatocopherol deposition across laying order varied with mate attractiveness (Figs 2 and 3). This result was not due to differences in egg size or the proportion of yolk, as neither differed with either laying order or treatment. Also, there was a significant interaction between mate attractiveness, laying order and the total amount of carotenoid and alpha-tocopherol in whole yolk. Zebra Finches hatch asynchronously (Zann 1996). In species with asynchronous hatching, eggs hatch in the order of laying, resulting in an age size hierarchy within the brood. Later-hatched chicks typically show a slower growth rate (Lago, Johnson & Albrecht 2000; Johnson et al. 2003) and higher mortality (Graves, Whiten & Henzi 1984; Mock, Drummond & Stinson 1990) than those hatching from earlier-laid eggs. By depositing relatively more antioxidants in later-laid eggs when paired to an attractive male, females may increase the survival prospects for later-hatched chicks and the probability of a greater number of offspring surviving to adulthood. This investment may be worthwhile as, assuming the offspring will themselves be more attractive, it increases the lifetime reproductive success of the laying female. It should be noted, however, that whereas the effect of differing yolk antioxidant concentrations on chick fitness has been measured for domesticated species (Surai 2002), further studies are necessary to define the relationship between individual antioxidant concentration and chick fitness in the Zebra Finch. For carotenoids, the pattern of concentration with laying order shows that only the last egg in clutches

358 K. A. Williamson et al. laid for attractive males contains higher concentrations. However, for alpha-tocopherol, concentration with laying order is greater from the third-laid egg onwards. This is particularly interesting as alpha-tocopherol has a far greater antioxidant potential than that of carotenoids (Halliwell & Gutteridge 1999; Surai 2002). Female body condition measured as fat score had a significant effect on the concentration of carotenoids in yolk (Fig. 1) but not on alpha-tocopherol. This suggests that females in enhanced condition, with greater fat reserves, are more capable of provisioning eggs with carotenoids. Whereas alpha-tocopherol is stored mostly in the liver, carotenoids are also stored throughout the body in several areas including the integument and body fat. How could this differential provisioning be achieved? Yolk formation is under the control of plasma oestrogen levels (Johnson 2000). Yolk is synthesized in the liver and then transported via the blood to the developing follicle. Lipid-soluble antioxidants stored in the liver are thought to be incorporated in yolk within the liver and subsequently deposited in the follicle (Surai 2002). Oestrogen levels peak at about the time the first egg of a clutch is ovulated (Bahr et al. 1983), then decline as more eggs are laid, reaching a minimum with the ovulation of the last egg. This decrease in oestrogen over the laying sequence reflects the declining levels of alpha-tocopherol and carotenoids found in the eggs with laying order. Oestrogen may influence the concentration of lipid-soluble antioxidants in egg yolk. Oestrogen is known to have an antioxidant effect, preventing peroxidation of lipids (Halliwell & Gutteridge 1999), and may have a role in regulating the amount of vitamin E in the liver (Feingold, Longhurst & Colby 1993). Poultry administered with oestrogen showed higher plasma levels of vitamin E (Halifeoglu et al. 2003). Several other hormones are also active during oogenesis, and subtle interactions exist between them (Johnson 2000). Hormones almost certainly influence the level of antioxidants deposited in the developing follicle (Surai 2002), but the identity of the hormone responsible remains to be determined. Environmental factors influence female hormone levels during reproduction. Several studies have found a relationship between competition, aggression and levels of testosterone in egg yolk (Schwabl 1996; Whittingham & Schwabl 2002). Mate attractiveness has also been shown to affect levels of circulating hormones and those in yolk. Gwinner, Van t Hof & Zeman (2002) found that concentrations of luteinizing hormone in female European Starlings (Sturnus vulgaris) were higher when paired with a higher-quality male. Female Canaries (Serinus canaria) and Zebra Finches deposit more testosterone in eggs laid for attractive males (Gil et al. 1999; Gil et al. 2004; Tanvez et al. 2004). These studies suggest that having an attractive mate may prompt a physiological reaction, which may ultimately lead to the manipulation of egg composition. Acknowledgements We thank Graham Kemp for his advice on HPLC, Filiz Karadas for help with the antioxidant extractions, and also Lucy Gilbert and Monique Mackenzie for advice on the statistical analysis. References Andersson, M. (1992) Sexual Selection. Princeton University Press, Princeton, NJ, USA. Bahr, J.M., Wang, S.C., Huang, M.Y. & Calvo, F.O. (1983) Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen. Biology of Reproduction 29, 327 334. Blount, J.D., Surai, P.F., Nager, R.G. et al. (2002) Carotenoids and egg quality in the lesser black-backed gull Larus fuscus: a supplemental feeding study. Proceedings of the Royal Society of London Series B 269, 29 36. Blount, J.D., Houston, D.C., Surai, P.F. & Moller, A.P. (2004) Egg-laying capacity is limited by carotenoid pigment availability in wild gulls Larus fuscus. Proceedings of the Royal Society of London Series B 271, S71 S81. Burley, N. (1988) The differential-allocation hypothesis: an experimental test. American Naturalist 132, 611 628. Burley, N., Krantzberg, G. & Radman, P. (1982) Influence of colour-banding on the conspecific preferences of zebra finches. Animal Behaviour 30, 444 455. Cunningham, E.J.A. & Russell, A.F. (2000) Egg investment is influenced by male attractiveness in the mallard. Nature 404, 74 76. Feingold, I.B., Longhurst, P.A. & Colby, H.D. (1993) Regulation of adrenal and hepatic alpha-tocopherol content by androgens and estrogens. Biochimica et Biophysica Acta 1176, 192 196. Gil, D., Graves, J., Hazon, N. & Wells, A. (1999) Mate attractiveness and differential testosterone investment in zebra finch eggs. Science 286, 126 128. Gil, D., Leboucher, G., Lacroix, A., Cue, R. & Kreutzer, M. (2004) Female canaries produce eggs with greater amounts of testosterone when exposed to preferred male song. Hormones and Behaviour 45, 64 70. Grafen, A. & Hails, R. (2002) Modern Statistics for the Life Sciences. Oxford University Press, Oxford, UK. Graves, J., Whiten, A. & Henzi, P. (1984) Why does the herring gull lay three eggs? Animal Behaviour 32, 798 805. Gwinner, H., Van t Hof, T. & Zeman, M. (2002) Hormonal and behavioural responses of starlings during a confrontation with males or females at nest boxes during the reproductive season. Hormones and Behaviour 41, 21 31. Halifeoglu, I., Karatas, K., Canatan, H., Colak, R. & Karadas, E. (2003) Investigation of antioxidant vitamins (A, E and C) and selenium levels in chickens receiving estrogen or testosterone. Cell Biochemistry and Function 21, 133 136. Halliwell, B. & Gutteridge, J.M.C. (1999) Free Radicals in Medicine and Biology, 3rd edn. Oxford University Press, Oxford, UK. Haq, A., Bailey, C.A. & Chinnah, A. (1996) Effect of betacarotene, canthaxanthin, lutein, and vitamin E on neonatal immunity of chicks when supplemented in the broiler breeder diets. Poultry Science 75, 1092 1097. Helms, C.W. & Drury, W.H. (1960) Winter and migratory weight and fat field studies on some North American buntings. Bird Banding 31, 1 40. Johnson, A.L. (2000) Reproduction in the female. Sturkie s Avian Physiology (eds G. Causey Whitlow). Academic Press, London. Johnson, L.S., Wimmers, L.E., Campbell, S. & Hamilton, L. (2003) Growth rate, size, and sex ratio of last-laid, last-hatched

359 Yolk antioxidants and mate attractiveness offspring in the tree swallow Tachycineta bicolor. Journal of Avian Biology 34, 35 43. Lago, K., Johnson, L.S. & Albrecht, D.J. (2000) Growth of late-hatched, competitively disadvantaged nestling house wrens relative to their older, larger nestmates. Journal of Field Ornithology 71, 676 685. Lessells, C.M. (1991) The evolution of life histories. In: Behavioural Ecology: An Evolutionary Approach, 3rd edn (eds J.R. Krebs & N.B. Davies). Blackwell Scientific Publications, Oxford, UK. Mock, D.W., Drummond, H. & Stinson, C.H. (1990) Avian siblicide. American Scientist 78, 438 449. Mousseau, T.A. & Fox, C.W. (1998) Concluding remarks. In: Maternal Effects as Adaptations (eds T.A. Mosseau & C.W. Fox), pp. 345 348. Oxford University Press, Oxford, UK. Nager, R.G. Monaghan, P. & Houston, D.C. (2001) The cost of egg production: increased egg production reduces future fitness in gulls. Journal of Avian Biology 32, 159 166. Petrie, M. & Williams, A. (1993) Peahens lay more eggs for peacocks with larger trains. Proceedings of the Royal Society of London Series B 251, 127 131. Royle, N.J. Surai, P.F. & Hartley, I.R. (2001) Maternally derived androgens and antioxidants in birds eggs: complementary but opposing effects? Behavioral Ecology 12 (4), 381 385. Royle, N.J. Surai, P.F. & Hartley, I.R. (2003) The effect of variation in dietary intake on maternal deposition of antioxidants in zebra finch eggs. Functional Ecology 17, 472 481. Rutstein, A.N. Gilbert, L. Slater, P.J.B. & Graves, J.A. (2004) Mate attractiveness and primary resource allocation in the zebra finch. Animal Behaviour 68, 1087 1094. Saino, N. Dall ara, P. Martinelli, R. & Møller, A.P. (2002a) Early maternal effects mediated by immunity depend on sexual ornamentation of the male partner. Proceedings of the Royal Society of London Series B 269, 1005 1009. Saino, N. Bertacche, V. Ferrari, R.P. Martinelli, R. Møller, A.P. & Stradi, R. (2002b) Carotenoid concentration in barn swallow eggs is influenced by laying order, maternal infection and paternal ornamentation. Proceedings of the Royal Society of London Series B 269, 1792 1733. Schwabl, H. (1996) The contents of maternal testosterone in house sparrow Passer domesticus eggs vary with breeding conditions. Naturwissenschaften 84, 406 408. Sheldon, B.C. (2000) Differential allocation: tests, mechanisms and implications. Trends in Ecology and Evolution 15, 397 402. Stearns, S.C. (1992) The Evolution of Life Histories. Oxford University Press, Oxford, UK. Surai, P.F. (2002) Natural Antioxidants in Avian Nutrition and Reproduction. Nottingham University Press, Nottingham, UK. Tanvez, A. Beguin, L. Chastel, O. Lacroix, A. & Leboucher, G. (2004) Sexually attractive phrases increase yolk androgen deposition in Canaries (Serinus canaries). General and Comparative Endocrinology 138, 113 120. Veasey, J.S. Houston, D.C. & Metcalf, N.B. (2001) A hidden cost of reproduction: the trade-off between clutch size and escape take off-speed in female zebra finches. Journal of Animal Ecology 70, 20 24. Verboven, N. Evans, N.P. D Alba, L. et al. (2005) Intraspecific interactions influence egg composition in the lesser black backed gull (Larus fuscus). Behavioral Ecology and Sociobiology 57, 357 365. Whittingham, L.A. & Schwabl, H. (2002) Maternal testosterone in tree swallows varies with female aggression. Animal Behaviour 63, 63 67. Zann, R.A. (1996) The Zebra Finch. Oxford University Press, Oxford, UK. Received 2 August 2005; revised 10 October 2005; accepted 7 November 2005 Associate Editor: Gary Bortolotti