Yolk testosterone levels and dietary carotenoids influence growth and immunity of grey partridge chicks

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1 Available online at General and omparative Endocrinology 156 (2008) Yolk testosterone levels and dietary carotenoids influence growth and immunity of grey partridge chicks Marco ucco *, Beatrice Guasco, Giorgio Malacarne, Roberta Ottonelli, Aurélie Tanvez University of Piemonte Orientale, DISAV Dipartimento di Scienze dell Ambiente e della Vita, via Bellini 25, I Alessandria, Italy Received 3 October 2007; revised 18 December 2007; accepted 25 December 2007 Available online 18 January 2008 Abstract Early maternal effects in the form of substances accumulated in the egg, such as carotenoids and hormones, can be physiologically relevant for a good development of offspring. It has been found in different species that testosterone (T) can be beneficial to offspring by increasing growth rate, but detrimental by reducing immunocompetence and increasing oxidative stress. arotenoids on the other hand are suggested to be beneficial because they can counteract the oxidative stress and the immune-depressive effect of T. In this study we analyzed the effect of prenatal T exposure in the grey partridge. We injected eggs with three doses of T (high, intermediate, and physiological). After hatching, chicks exposed to a prenatal high dose of T were fed with two diets (rich or poor) differing in b-carotene content. We found a significant effect of T on both chick growth and cell-mediated immunity, with high T doses resulting in detrimental effects while low doses were beneficial. Detrimental effects of the high dose of T on immunity were mitigated by b-carotene consumed in the diet. The differences between groups were observed in the early period of life (age 10 days for mass, and age 10 and 21 days for immunity), and disappeared in the following period, and up to 1 and 2 years later. Overall, our observations show that T in the egg is not detrimental but beneficial, and that negative effects are found only at supraphysiological concentrations. The negative effects of T on immunity could be balanced if chicks could consume a diet rich in b-carotene. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Perdix perdix; Maternal effects; Testosterone; b-arotene; Egg-injection 1. Introduction Maternal effects represent a peculiar environmental source of phenotypic variance in early-life traits; for example in viviparous animals, it has been shown that they influence birth mass, birth date, mortality, etc. (Mousseau and Fox, 1998). Oviparous animals show specific early maternal effects in the form of substances accumulated in the egg, such as carotenoids, antibodies and hormones that can be physiologically relevant for a good development of offspring (Radder and Shine, 2007). In the last decade, from the early studies of Schwabl (1993), Ros et al. (1997), Royle et al. (1999) and Hasselquist et al. (1999) among others, increasing effort has been directed towards * orresponding author. Fax: address: marco.cucco@unipmn.it (M. ucco). the physiological effects of these maternally derived substances. oncerning sexual hormones, it has been shown that yolk androgens stimulate growth in many species (Groothuis et al., 2005). This effect could be related to their general anabolic properties, or be mediated by an enhanced competitive capability to obtain food from parents and aggressive behaviour during growth (Schwabl, 1996; Lipar and Ketterson, 2000; Lipar, 2001; Eising and Groothuis, 2003; Groothuis and Ros, 2005; Goodship and Buchanan, 2006). However, other studies of prenatal exposure to elevated T levels in ovo on post-hatching growth, have provided nil or even negative evidence (Groothuis et al., 2005; Boncoraglio et al., 2006). Besides the effect on growth, yolk T is supposed to have costs since elevated T levels are associated with high oxidative stress and immunosuppressive effects (Foldstad and Karter, /$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi: /j.ygcen

2 M. ucco et al. / General and omparative Endocrinology 156 (2008) ; Duffy et al., 2000; Anderson et al., 2004; Navara et al., 2005). In line with the effects on growth, the evidence of a negative effect of T on immune response is far from unequivocal. In most studies the depressive effect was clear (Peters, 2000; Eising et al., 2001; Eising and Groothuis, 2003; Müller et al., 2005b; Navara et al., 2005), but in some cases T injection did not influence the immunity (Anderson et al., 2004; Tschirren et al., 2005; Rubolini et al., 2006b) and, in a recent paper, even a positive effect of T egg-injection on chicks immunity has been reported (Navara et al., 2006b). Discussing the effects of embryonic T as a maternal effect, Rubolini et al. (2006b) consider that the consequences of elevated yolk T in birds can differ even between closely related taxa, and the effects of yolk androgens on growth and development are species-specific (Navara et al., 2006b). Androgens deposited into the yolk can have a variety of effects because of physiological differences between the offspring of different species, and even within the same species opposite effects of yolk testosterone on sexes have been found (von Engelhardt et al., 2006). It is thus clear that analyses of the species-specific costs and benefits associated with the strategies of yolk androgen distribution are necessary before we can understand their adaptive significance (Navara et al., 2006b). It is also clear that multi-factorial models of the maternal effects must be proposed to explain the heterogeneous and apparently contradictory data (Navara et al., 2006a). A major point in this field is that substances accumulated in the egg are expected to benefit the chick, but results from manipulative experiments are contrasting (Blount et al., 2000; Groothuis et al., 2005), and mother-offspring tradeoff and pleiotropic interactions between different egg substances may play an important role (Royle et al., 2001). A first variable to be considered in experimental studies is the quantity of substances (hormones) injected. In a review of the function of maternal hormones in avian species, Groothuis et al. (2005) caution against the use of high supraphysiological doses, because the functional consequences of such a treatment may become difficult to interpret. However, in some species a dose of androgens beyond physiological range was utilized (Schwabl, 1996; Anderson et al., 2004; Pilz et al., 2004; Navara et al., 2005) and was demonstrated to be beneficial for Bluebird Sialia sialis and Starling Sturnus vulgaris growth (Pilz et al., 2004; Navara et al., 2005), and detrimental for hinese painted quail oturnix chinensis and Bluebird immune function (Anderson et al., 2004; Navara et al., 2005). To better understand the importance of the quantity of androgen administered, in the present study we employed both physiological and supraphysiological doses. The interactions of egg substances play an even more important role. It is supposed that egg carotenoids are accumulated to protect from the negative effects of T, since these substances are beneficial to embryonic immunity and to protection from oxidative stress (Royle et al., 2001). Indeed, an experimental injection of the lutein carotenoid led to improvement of immunocompetence in the Barn swallow Hirundo rustica chicks (Saino et al., 2003). If egg yolk androgen and antioxidants are both adaptive maternal effects, we would expect that the two maternal effects can interact in different ways. A first possibility is that their benefits are additive, thus providing a mechanism for parental intraclutch favouritism or, on the contrary, for a brood survival strategy if substances are allocated preferentially to the last hatched nestling to compensate for hatching asynchrony disadvantage (Török et al., 2007). On the other hand, the benefit provided by T and carotenoids could act in an opposite way in egg, as found in the Lesser black-backed gulls Larus fuscus, where an opposite concentrations of T and carotenoids were found in the three-egg clutch (Royle et al., 2001). Moreover, the possibility should be taken into account that the quantity of these two substances allocated in the eggs could not be optimal for the embryo because the evolutionary interests of the mother and offspring differ (Müller et al., 2007). The effects of precocial exposure to carotenoids has rarely been studied by egg-injection experiments (Saino et al., 2003), the commonest technique employed being experimental nutrition of new hatched young with diets differing in carotenoid content. Dietary experiment showed the existence of a general beneficial effect of these substances to growth and immunity (Surai, 2002; Fenoglio et al., 2002; ucco et al., 2006a). In this study we analyzed the effect of embryonic T exposure in the Grey partridge Perdix perdix. In order to make clear the role of T for chick development we used three doses of T (high, intermediate, and physiological). Since carotenoids may counteract the immune-depressive and oxidant role of T, the chicks exposed to a high dose of T were fed with two diets (rich or poor) differing only in b-carotene content. hick body mass and cell-mediated immunity were the two parameters studied in this research. 2. Methods 2.1. Study area and experimental design The study was conducted on grey partridges reared in 2003, 2005 and 2006 at a game breeding farm in S. Giuliano Nuovo, Alessandria, NW Italy (ucco et al., 2006b, 2007). In total, 14 breeding pairs in 2003, 23 in 2005 and other 18 pairs in 2006 were housed in individual outdoor reproduction cages (4 m long 1 m wide 0.5 m high). Throughout the year, the birds were maintained in natural light and temperature conditions. The hens laid a total of 564 eggs in 2003, 855 eggs in 2005, and 289 eggs in Freshly laid eggs were individually marked with a non-toxic marker to record parental identity and the date of laying, and then incubated for 26 days in a commercial incubator at 37.5 and 60% humidity. After hatching, chicks were individually tagged with a numbered plastic ring on the leg to allow subsequent identification. The influence of testosterone on chick health and body condition was investigated by egg-injection (Fluka testosterone). Eggs were injected after laying and immediately prior to incubation with one of three injection treatments: (1) High-dose, 20 lg T in 20 ll sesame oil, N = 69 eggs in 2003; (2) Middle-dose, 200 ng T in 20 ll sesame oil, N = 112 eggs in 2005; (3) Low-dose, 20 ng T in 20 ll sesame oil, N = 83 eggs in Each year, we also created a control group of eggs injected with 20 ll sesame oil (N = 41, 95, and 84 eggs in 2003, 2005, and 2006, respectively). Egg were assigned to treatment groups by distributing eggs from breeding pairs equally across groups.

3 420 M. ucco et al. / General and omparative Endocrinology 156 (2008) The T injection amounts were chosen based on 18 grey partridge eggs collected from the study breeding farm in 2002, which were found to vary from 16.2 to 25.5 ng/yolk, with a mean concentration of 3.38 pg/mg (unpublished data, yolk concentration of T determined by radioimmunoassay, courtesy of Prof. oncetta Lupo, University of Siena, Italy). Eggs were wiped with 70% ethanol, and injections were performed using a 25 ll Hamilton syringe mounting a 22-gauge needle. To test whether the vehicle containing the treatments actually reached the yolk, we injected 20 ll of sesame oil stained with Sudan B into 15 extra eggs. The yolks of all of these eggs were ascertained to contain the stain after shell breakage. The influence of dietary carotenoids on chick health and body condition was investigated in 2003 by b-carotene supplementation to chicks. We chose b-carotene because of its known effect on immune condition (Tengerdy et al., 1990; Haq et al., 1995). b-arotene is efficiently converted to vitamin A (Moren et al., 2002). However, since we did not use enzyme blockers or radio-labelled and tracked carotenoids to investigate the direct molecular effects, we do not know the active molecular form in the partridge body. We assigned chicks to two groups (fed with low or high b-carotene food). hicks in the low b-carotene group received a standard chick partridge diet of cereal pellet plus 2.2 mg/kg of b-carotene, while the high b-carotene group received the same standard food plus 22 mg/kg of b-carotene. The high b-carotene level was chosen to match the value usually used in Italian grey partridge breeding farms, near the highest value utilized in poultry, with a high safety margin with respect to the NR (National Research ouncil) recommendation. The low b-carotene level was 10 times lower than the high level, near to the minimum nutrient requirement reported by NR recommendation (Villamide and Fraga, 1999; NR, 1994). The rearing food was a powdered mixture commonly used by aviculturists to provide proper nutrition during egg laying (nutrition facts: protein 19.5%, fat 3.7%, ash 11.5%). The mixture of food and b- carotene was calibrated and supplied by Mucedola srl, Settimo Milanese, Italy. Food and fresh water were always available. hicks of the different b-carotene experimental groups were raised for six weeks in separate heated pens in a nursery room maintained at 20.5 ± 2.0. After six weeks, the chicks were removed from the heated pens and placed in large outdoor aviaries Growth and immunological test We measured the body mass of all chicks with an electronic balance (±0.01 g accuracy) at 10, 21 and 90 days of age. ell-mediated immunity was assessed on three occasions, at age 10, 21 and 90 days. We used the PHA test to measure the delayed cutaneous hypersensitivity response to injection of phytohaemoagglutinin (PHA), a foreign antigen that causes T-lymphocyte proliferation and local swelling (Lochmiller et al., 1993). For each chick, a small area on the right wing web (patagium) was marked with non-toxic ink. The thickness of the right wing web was measured with a thickness gauge with an accuracy of 0.01 mm. The right wing web area was then injected intradermally with 0.25 mg of PHA (Sigma L-8754) dissolved in 0.05 ml of phosphate-buffered saline (PBS). After 24 h, the wing web thickness in the marked area was re-measured. The subcutaneous injection with PHA produces local inflammation, and the increased wing web thickness is directly related to the immunological condition (Smits et al., 1999). A subset of animals hatched in 2005 was kept in the aviaries in order to test long-term effects of treatment. Their mass and PHA reaction was measured in the first year of life at day 260, and 2 years after hatching at day 625. Moreover, at days 260 and 625 we measured their blood erythrosedimentation rate and haematocrit Statistics The effect of T treatment on body mass, response to PHA, erythrosedimentation rate and haematocrit was analyzed using linear mixed models (LMM procedure in SYSTAT 12, Wilkinson 2007), with year, injection treatment and year treatment interactions as fixed effects. Because each female laid several eggs, characteristics of hatched siblings may not be independent. Hence, to control for the potential effects of parents in this experiment, we inserted the parent identity as a random effect in all the analyses. Erythrosedimentation rates were arcsine transformed before analysis. The effect of b-carotene treatment was analyzed using mixed models, with b-carotene and T treatment as fixed effects, and parent identity as a random effect. The year was not inserted in this analysis, because the different diets were provided to chicks only in Results 3.1. Effects of testosterone injection The effect of T treatment on mass varied significantly in relation to the year, and there was also a significant year T treatment interaction (Table 1). The effect of T was positive in the low-dose group, and negative in the high-dose group at age 10 days. Post hoc comparisons indicated that chicks hatching from eggs that were injected with the high-dose treatment showed a significantly lower mass than control chicks (Fig. 1); F 1,13 = 4.87, P < On the contrary, low-dose chicks were significantly heavier than chicks in the control group (F 1,25 = 6.98, P < 0.014), and middle-dose chicks did not significantly differ from control group chicks (F 1,31 = 0.28, P = 0.60). Mass differences among treatment groups disappeared by days 21 and 90 (days 21: Table 1; day 90: F 1,11 = 0.105, P = 0.75). The effect of T treatment on immune response varied significantly in relation to the year, and there was also a significant year T treatment interaction (Table 1). The effect of T was positive in the low-dose group, and negative in the high-dose group at age 10 days. Post hoc comparisons indicated that chicks hatching from eggs that were injected with the high-dose treatment showed a significantly lower immune response than control chicks (Fig. 2); F 1,13 = 4.92, P < On the contrary, low-dose chicks had a significantly higher immune response than chicks in the control group (F 1,25 = 10.05, P < 0.004), and Table 1 Mixed model ANOVAs for body mass and immune reaction in relation to T treatment and year (fixed effects) Dependent variable Factors d.f. F P Mass at age 10 T treatment 2, Year 1, Year T treatment 2, Mass at age 21 T treatment 1, Year 2, Year T treatment 2, Immune reaction at age 10 T treatment 1, Year 2, Year T treatment 2, Immune reaction at age 21 T treatment 1, Year 2, Year T treatment 2, In the models, parent identity was entered as a random effect factor linking chicks from the same parents.

4 M. ucco et al. / General and omparative Endocrinology 156 (2008) Mass at age 10 (g) P < P = 0.60 P < middle-dose chicks did not significantly differ from control group chicks (F 1,29 = 0.01, P = 0.95). Immune response differences among treatment groups was still present at age 21 days (Table 1), but disappeared by day 90 (F 1,11 = 0.31, P = 0.59). The erythrosedimentation rate and the haematocrit were not assessed in chicks hatching from eggs that were injected with the high-dose treatment, but only in chicks pertaining to the middle-dose, low-dose, and control groups. There was no long-term effect of T on haematocrit and erythrosedimentation rate among the treatment groups; they were similar both on day 260 (haematocrit F 2,28 = 0.33, P = 0.74; ES rate F 2,34 = 1.84, P = 0.11) and on day 625 post-hatch (haematocrit F 1,5 = 1.26, P = 0.31; ES rate F 1,5 = 0.80, P = 0.41). Moreover, there was no long-term effect of injection on mass and immune response among the treatment groups; they were similar both the following year (day 260 mass: Fig. 1. The effects of in ovo testosterone and control injection treatments on mean (SD) mass of chicks 10 days after hatching. Injection treatments included a control injection (20 ll sesame oil), a high-dose injection (20 lg T in 20 ll sesame oil), a middle-dose injection (200 ng T in 20 ll sesame oil) and a low-dose (20 ng T in 20 ll sesame oil). Immune response at age 10 (mm) P < P = P < Highdose Middledose Lowdose Highdose Middledose Lowdose Fig. 2. The effects of in ovo testosterone and control injection treatments on mean (SD) immune response of chicks 10 days after hatching. Injection treatments included a control injection (20 ll sesame oil), a high-dose injection (20 lg T in 20 ll sesame oil), a middle-dose injection (200 ng T in 20 ll sesame oil) and a low-dose (20 ng T in 20 ll sesame oil). F 2,35 = 3.05, P = 0.06; immune response F 2,34 = 1.25, P = 0.30) and 2 years later (day 625 mass: F 1,5 = 4.38, P = 0.09; immune response: F 1,5 = 0.01, P = 0.98) Effects of b-carotene nutrition The effects of b-carotene nutrition were examined only on chicks hatching from eggs that were injected with the high-dose T treatment and with the control treatment. There was no effect of b-carotene nutrition on mass both at age 10 and age 21 (Table 2). Regardless of the diet, at age 10 there was a significant difference in mass in relation to T treatment, with control chicks being heavier than chicks hatched from eggs injected with the high dose. No effect of T treatment was detectable at age 21 (Table 2). At age 10 days, chicks fed with a high b-carotene content in their diet showed a significantly higher immune reaction than chicks fed with a low b-carotene diet, regardless of the T treatment (Fig. 3A; Table 2). An immune response difference among b-carotene groups was still present by day 21 (Fig. 3B; Table 2). There was a significant interaction between diet and T treatment, i.e. there was a negative effect of T treatment on immune reaction of chicks fed with a high b-carotene diet (age 10, Fig. 3A: F 1,13 = 4.92, P < Age 21, Fig. 3B: F 1,13 = 4.97, P < 0.044), while T treatment had no effect on the chicks fed with a low b-carotene diet (age 10, Fig. 3A: F 1,12 = 0.48, P = Age 21, Fig. 3B: F 1,12 = 0.87, P = 0.37). 4. Discussion In this study we experimentally increased in ovo concentrations of yolk T. We found a significant effect of T on both chick growth and immunity, with a high T dose resulting in a detrimental effect while low dose was beneficial. The detrimental effects of a high T dose were mitigated by b-carotene consumed in the diet. Table 2 Mixed model ANOVAs for body mass and immune reaction in relation to T treatment and b-carotene diet (fixed effects) Dependent variable Factors d.f. F P Mass at age 10 T treatment 1, Diet 1, Diet T treatment 1, Mass at age 21 T treatment 1, Diet 1, Diet T treatment 1, Immune reaction at age 10 T treatment 1, Diet 1, Diet T treatment 1, Immune reaction at age 21 T treatment 1, Diet 1, Diet T treatment 1, In the models, parent identity was entered as a random effect factor linking chicks from the same parents.

5 422 M. ucco et al. / General and omparative Endocrinology 156 (2008) Immune reaction at age 10 (mm) Immune reaction at age 21 (mm) Sesame 5 P < P < High dose T P < 0.05 β low β high β low β high Sesame P < β-carotene diet P < High dose T P < β low β high β low β high β-carotene diet Fig. 3. The effects of in ovo testosterone and b-carotene supplementation to chicks on mean (SD) immune response of chicks 10 days and 21 days after hatching. Injection treatments included a control injection (20 ll sesame oil) and a high-dose injection (20 lg T in 20 ll sesame oil). The b- carotene supplementation included a b-low (2.2 mg/kg) and a b-high (22 mg/kg) chick food. In our study, the injection of a high dose of T induced a reduction of chick mass, while the physiological dose increased the mass in comparison to control. An increase of yolk T, in general, promotes growth of nestlings (a few exceptions are reported in Sockman and Schwabl, 2000; Rubolini et al., 2006c; rev. in Groothuis et al., 2005) suggesting that maternal investment via deposition of yolk T could promote fitness-related growth of nestlings. oncerning metabolic mechanisms, androgens have been found to stimulate the release of bone growth factors as well as cartilage cell proliferation in mammals (orvol et al., 1992; Fischer et al., 1995). Androgenic hormones have well-known anabolic effects, causing nitrogen retention and increased muscle mass due to hypertrophy of myofibrils (Mooradian et al., 1987). Yolk injections have shown that T has a immunosuppressive effect in most species studied to date (rev. Groothuis et al., 2005). This finding is in line with the wellknown immunosuppressive effect of T in vertebrates (Foldstad and Karter, 1992; Wedeking and Folstad, 1994). Our study contradicts this generally supported finding, since injections of T at physiological doses elevated cell-mediated immunity. There are a few other studies that cast doubts on a per se immunosuppressive effect of T. A positive effect of prenatal T exposure on immunity was found in a passerine, the house finch (Navara et al., 2006b), and in the Siberian hamsters Phodopus sungorus (Bilbo and Nelson, 2001). Interestingly, in accord with an immune-enhancing androgen effect, egg treatment with an anti-androgen (flutamide) was related to a reduction of immunity, and not to an immunity advantage as immunosuppressive theory would predict (Müller et al., 2005a). aution however must be used before definitely defining as beneficial the immunological effects of yolk T in our species. It is possible that while yolk androgens had a stimulatory effect on the cell-mediated immune response, the same treatment might have a different effect on other aspects of immunity, like antibody-mediated immune response and the innate phagocytic response (Navara et al., 2005). Moreover, our study was conducted in an optimal environmental situation, in absence of food-shortage, because growing chicks were fed ad libitum. The effect of T could be contextdependent: when food is abundant it is likely that the energy trade-off that might normally exist between growth and immunity in developing offspring is absent (Navara et al., 2006a; Pérez-Rodriguez et al., 2006). In our study, the T showed a positive effect when injected at a low dose, while the positive effect disappeared at middle-dose, and was replaced by a negative effect at high, supra-physiological dose. A dose-dependent effect is quite evident. Even if Groothuis et al. (2005) caution against the use of high doses, because the functional consequences of such a treatment may become difficult to interpret, we think that in our study the multiple dose experiment led to some interesting considerations. Pilz et al. (2004), using a dose of T beyond physiological range, showed that the chick mass of Starlings benefited from the unnaturally high T levels. The authors suggested that the effects observed probably represent physiological not pharmacological androgenic effects because Starling eggs contain relevant quantities of other androgens (A4 and DHT) so that the high T dose used reflects a physiological dose of total androgens. High T doses have also been demonstrated beneficial for Bluebird chick s growth (Navara et al., 2005), but detrimental for Bluebird and hinese painted quail immune function (Anderson et al., 2004). In our study, the middle dose of T had no effect on mass and immunity. Indeed, both immunity and mass of the middle-dose group did not differ from the control group. This is an intermediate outcome in comparison to the positive effect of the low dose, and to the negative effect of the high dose of T. However, one should be cautious before concluding that the middle-dose corresponds exactly to the dose of transition from a positive vs. a negative effect. There were year to year differences in our experimental condition, and the experiment with the middle-dose of T was performed in a year when the immune reaction was

6 M. ucco et al. / General and omparative Endocrinology 156 (2008) low also in the control group. It is possible that, in a more favourable year, a difference between middle-t and control groups could be evidenced. This aspect could be better explained in future experiments employing several different doses of T in the middle range comprised between the low and the high dose, and in long-term studies elapsing on different years (Acquarone et al. 2002). In this study we have shown that the negative effects, both on growth and immunity, of a high dose of T in ovo are reduced in the chick group fed with a rich b-carotene diet. The immune suppressive effect was not observed when chicks already had a reduced immune reaction, due to a poor b-carotene diet. Dietary and egg-injection experiments showed the existence of a general beneficial effect of carotenoids on growth and immunity (Surai, 2002; Saino et al., 2003; ucco et al., 2006a). To our knowledge this is one of the first experimental studies verifying the assumption that T (high dose) and carotenoids can have complementary and opposing effects (Royle et al., 2001). The relevance of carotenoids in the yolks of eggs have been examined mainly in descriptive studies concerning the pattern of deposition of yolk testosterone in eggs within clutches. For example, in the Black-backed gull it has been suggested that these substances can have balancing effects, perhaps resulting from high levels of oxidative stress experienced by chicks in relation to the contents of the eggs from which they hatched (Royle et al., 2001, see also Ruolini et al., 2006a; Török et al., 2007). Also, in the ollared flycatcher Ficedula albicollis, yolk testosterone is positively related to b-carotene content. Females laying eggs with a high testosterone concentration allocate more b-carotene to their eggs, possibly to counteract the potentially detrimental effects of steroids (Török et al., 2007). However, it has been suggested that there are carotenoids that stimulate the production of steroid hormones (Blas et al., 2006). Testosterone treatment in adult Red-legged partridge Alectoris rufa increases the bioavailability of carotenoids in plasma and liver. Plasma carotenoids, in turn, could be responsible for individual differences in immune response (Blas et al., 2006). A synergism of the two substances is probably at work, but more studies are needed to clarify this point (see also Safran et al., 2007). In our study species, we found that the positive effects of T and b-carotene on growth disappeared with the development of the birds. A similar trend was already found in a previous experiment concerning b-carotene only (ucco et al., 2006a). The period of early growth is a critical time for the survival of partridge offspring (Browne et al., 2006) and it is conceivable that the positive effect can be critical in a long-term perspective. The fact that size measures were similar between control and T chicks by day 21 post-hatch, and still 1 and 2 years later, perhaps is not surprising because the chicks were reared in an optimal environment, without food restrictions. Possibly, when food is more limited, offspring that are larger early in the development period would outcompete the smaller offspring (Navara et al., 2006b). If a moderate increase of yolk T is indeed beneficial for offspring, then if possible females should allocate more T to eggs. learly, information is needed on the costs and benefits of egg T not only on a short-term but also on a longterm perspective. In subadult Grey partridges a behavioural benefit of T has been reported, since T implanted birds were more vigilant than controls when exposed to a predator (Fusani et al., 1997). Another case of a positive effect of T is reported for the House sparrow. Experimental manipulation of T in Sparrow eggs resulted in males having a large badge which is a sexually selected trait (Strasser and Schwabl, 2004). In addition, individuals were more likely to prevail in dominance relationships at a food source compared to individuals originating from control eggs. ases of long-term negative effects of prenatal T exposure are reported for the Pheasant Phasianus colchicus and the hinese painted quail. chinensis. In the Pheasant, yolk T injection resulted in a reduced length of male tarsal spurs, a trait which positively predicts viability and male success in intra and intersexual selection (Rubolini et al., 2006c). In the hinese painted quail, yolk T inoculation resulted in reduced testes mass (Uller et al., 2005). In summary, this study shows that T in the egg could enhance both growth and immunity in the early period of life. The b-carotene in the diet can improve chick s immune response in this critical period. The positive effect disappeared in the following period, and up to 2 years later. Studies examining the allocation of carotenoids and yolk hormones may be relevant steps toward uncovering the mechanism and the adaptive significance behind the distribution of these substances in the eggs. Acknowledgments We thank E. antone, G. De Vito, L. Borasi and A. Russo for help in the field work, Prof. oncetta Lupo for testosterone assay, and V. Garcia-Fernandez for useful comments on the manuscript. This study was supported by ATF Alessandria and 60 MURST grants. The study was conducted in the breeding farm owned by Dr. G. 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