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Genetic differences in yolk testosterone levels influence maternal hormone deposition in the second laying cycle in Japanese quails
Comparative Biochemistry and Physiology, Part A 164 (2013) 271–275
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Genetic differences in yolk testosterone levels influence maternal hormone deposition in the second laying cycle in Japanese quails Michal Zeman a, b,⁎, Peter Skrobanek b, Monika Okuliarova a a b
Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic
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Article history: Received 8 August 2012 Received in revised form 20 October 2012 Accepted 20 October 2012 Available online 25 October 2012 Keywords: Genetic variability Induced moult Maternal effects Quail Yolk testosterone
a b s t r a c t 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. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The egg represents the only source of nutritive and biologically active substances that the avian embryo can utilize for its growth and development. Egg composition has been extensively studied in relation to the functional role of essential nutrients such as proteins, lipids, vitamins (Finkler et al., 1998; Royle et al., 1999) and especially the considerable amounts of information carrying compounds transferred into the egg (Schwabl, 1993; Wilson and McNabb, 1997; Grindstaff et al., 2003). Maternal hormones have attracted probably the most attention since they represent a pathway for mediating trans-generational maternal effects. It is thought that the mothers may via hormone deposition into the egg transmit information about the post-hatching environment to the embryo and optimize the development and behaviour of their progeny (Groothuis et al., 2005). The maternal deposition of yolk androgens varies in relation to the environment experienced by the laying female (Gil, 2008) but fluctuations of social and ecological factors themselves are insufficient to explain high inter-female differences in yolk androgen levels documented under stable social and environmental conditions (Okuliarova et al., 2009). Recent studies have proved the genetic component of maternal hormone transfer into the egg on the base of mother–daughter ⁎ Corresponding author at: Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University Bratislava, Mlynska dolina, 842 15 Bratislava, Slovak Republic. Tel.: +421 2 602 96 424. E-mail address: [email protected] (M. Zeman). 1095-6433/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2012.10.028
resemblance in wild populations of the collared flycatcher, Ficedula albicollis (Tschirren et al., 2009) and a long-term selection experiment in the precocial Japanese quail, Coturnix japonica (Okuliarova et al., 2011a). The heritable variation of yolk androgen concentrations can account for nearly half of a total phenotypic variance of this trait (Okuliarova et al., 2011a) and it is essential for an understanding of evolutionary consequences of hormone-mediated maternal effects (Mousseau and Fox, 1998). Besides the high inter-individual variability in maternal hormone deposition marked intra-individual differences have been demonstrated in several avian species (Groothuis et al., 2005). In particular, altricial birds can produce a distinct pattern of yolk androgen concentrations within a single clutch (Schwabl, 1993; Eising et al., 2001) as well as between successively laid clutches (Schwabl, 1997; Gil et al., 2006; Tobler et al., 2007). This between-clutch pattern of yolk androgen deposition was not investigated in free-living precocial birds yet but a decline of yolk testosterone (T) concentrations was found between the early and the latest stages of the first reproductive cycle in domestic species (Okuliarova et al., 2011a; Guibert et al., 2012). Regarding this intra-individual variability, limited studies deal with changes of maternal hormone deposition across the longer period of the reproductive lifespan and in association with age-specific reproductive success that can be important from both the evolutionary and ecological perspectives. In domestic fowls, the duration of the egg producing period is considerably extended although both the egg laying intensity and egg quality gradually decrease after the first year of age (Woodard et al.,
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1973; Joyner et al., 1987). To restore reproductive performance to its former levels, egg producers induce moulting in their flocks applying short-term starvation and reducing the photoperiod. Such induced moulting results in the cessation of egg laying and reproductive quiescence for a limited period (a couple of weeks), which is followed by the second reproductive cycle (Berry, 2003). In the present study, we investigated whether maternal hormone deposition into the eggs varies between the first and the second laying cycles in precocial Japanese quail. We used genetic lines divergently selected for high (HET line) and low (LET line) egg T contents that can provide a useful model to study sources of yolk androgen variability represented by both the genetic and environmental component and genotype–environment interactions. To address how the genetically determined differences in maternal hormone deposition relate to female's breeding quality across two laying cycles we measured yolk and plasma T concentrations and egg quality traits in the HET and LET females at the end of the first and at the beginning of the second laying cycle after forced moulting. 2. Material and methods 2.1. Animals and moulting procedure Adult Japanese quail (C. japonica) from the fourth generation of the LET and HET lines divergently selected for yolk T concentrations (Okuliarova et al., 2011a) were used in the study. At the age of 46 weeks 16 pairs (8 LET and 8 HET) were taken from the original breeding population. Each female was housed with a familiar male from the same line in an individual cage (0.22 m 2). These pairs were kept stable for at least 4 weeks before starting the experiment. All birds were maintained in the same room at controlled temperatures (22 ± 2 °C) under a stimulatory photoperiod of 14 h of light (L) and 10 h of dark (D) with food and water ad libitum. For 2 weeks, daily egg production was recorded and the mean ± standard error of mean (SE) laying rates were 93.3 ± 4.7% and 85.6 ± 3.4% for the LET and HET females, respectively. At the end of this period the last 2 eggs per female were collected for yolk T analysis. A standard moulting procedure was applied to stop the laying and induce the second cycle (Berry, 2003). Females were placed into group cages according to the line and exposed to an inhibitory photoperiod of 8L:16D for 14 consecutive days. Food was removed for the first 24 h and for the rest of the treatment food and water were provided ad libitum. During the short days 6 LET and 7 HET females stopped laying and were moulting heavily. First, the feathers on both sides of the breast were lost, followed by secondary and primary reminges. The quails were then returned to their home cages with their males and re-exposed to the stimulatory photoperiod of 14L:10D. Twelve females (6 LET and 6 HET) restored regular egg production within 4 weeks and only eggs from those females were used for statistical analyses. Four weeks after the re-exposure to the stimulatory photoperiod, the eggs were collected for analyses across 6 days when the mean (±SE) laying rate was the same for both lines (83.3± 4.4%). The care and use of animals was in accordance with laws and regulations of the Slovak Republic and approved by the Ethical Committee of the Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic.
albumen, were thoroughly homogenized and stored at − 20 °C until steroid extraction and yolk T assay. Blood samples were obtained from the wing vein three times: 1) during the first laying cycle (39 weeks of age), 2) during the moulting (11 days after the shortening the photoperiod) and 3) during the second laying cycle (5 weeks after the re-exposure to the stimulatory photoperiod). Blood was collected in the morning, about 6–8 h before a predicted oviposition. Plasma was stored at −20 °C until T analysis. 2.3. Testosterone assay Testosterone was extracted from the yolk using the procedure described in detail by Okuliarova et al. (2009). Average recovery was 63.7± 0.7%. Testosterone concentrations were measured in both yolk extracts (20 μL aliquot) and plasma samples (50 μL aliquot) by radioimmunoassay using [1,2,6,7-3H]-T (Amersham Biosciences, UK, specific activity 3.52 TBq/mmol) and a specific antibody generated in rabbits against testosterone-3-(carboxy-methyl) oxime bovine serum albumin conjugate. The cross-reactivity of the antiserum was 9.6% with 5α-dihydrotestosterone, 0.1% with androstenediol and lower than 0.1% with other steroids (Zeman et al., 1986). Yolk and plasma samples were run separately within 4 assays with a mean intra-assay variation coefficient of 4% and inter-assay variation coefficient of 5.2%. The mean (±SE) assay sensitivity was 2.16 ± 0.16 pg/tube. 2.4. Statistical analyses The data for the yolk T levels and egg characteristics were averaged per individual female per laying cycle and analysed using repeated-
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2.2. Eggs and blood samples In total we collected 46 and 43 eggs from the LET and HET quails, respectively. Two eggs per female were analysed in the first laying cycle and 5.4 ± 0.2 (mean ± SE) eggs per female at the beginning of the second cycle. First, we measured the egg quality characteristics: egg mass, yolk mass and eggshell mass. The eggshell was weighed after cleaning and drying at 80 °C. The yolks, separated from the
Fig. 1. Plasma (A) and egg yolk (B) testosterone concentrations in the low (LET, N = 6) and high (HET, N=6) egg testosterone lines of Japanese quail in the first and the second laying cycle. Values represent means±SE. Different letters above the bars denote significant differences between groups (pb 0.05).
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measures analysis of variance with line as the between-group factor, laying cycle as the within-group factor and their interaction. Yolk T concentrations and total yolk T content were log transformed to fit the normal distribution and homogeneity of variance. All other data met these assumptions. Differences between groups were evaluated using Fisher's LSD post hoc tests. 3. Results The T concentrations in the female's plasma were not influenced by line (F1,10 = 0.14, p = 0.713), laying cycle (F1,10 = 1.09, p = 0.322) or the interaction between both factors (F1,10 = 0.70, p = 0.422) (Fig. 1A). During the period of forced moulting plasma T levels declined under the detection limit in all females (lower than 35 pg/mL) except for one that stopped laying eggs a few days after the blood sample collection. As expected mean yolk T concentrations in the egg were higher in the HET compared with the LET line (F1,10 = 29.14, p b 0.001). The effect of the laying cycle on yolk T concentrations was not significant (F1,10 = 2.47, p = 0.147) but a significant interaction between the line and laying cycle was found (F1,10 = 14.64, p b 0.01). Post hoc comparison revealed that yolk T concentrations had increased in eggs of the second laying cycle when compared with eggs of the first cycle in the HET quail (p b 0.01) while no significant change was found in the LET quail (Fig. 1B). Looking at the inter-female variation the majority of the LET quails showed a numerical decrease of yolk T deposition across two laying cycles (Fig. 2A). In contrast, all females except one in the HET line had increased yolk T levels in their eggs in the second laying cycle (Fig. 2B). The same pattern as for yolk T concentrations was found for total T content in the yolk (line: F1,10 = 36.21, p b 0.001; laying cycle:
F1,10 = 1.21, p = 0.298 and the interaction of line× laying cycle: F1,10 = 12.99, p b 0.01). The mean T levels (±SE) for the whole yolk were 22.1± 1.7 ng and 41.1± 3.7 ng at the end of the first cycle and 18.7 ± 2.1 ng and 60.8 ±10.2 ng at the beginning of the second cycle in the LET and HET lines, respectively. The second laying cycle did not affect either egg mass (F1,10 = 0.01, p = 0.935; Fig. 3A) or yolk mass (F1,10 = 0.11, p = 0.752; Fig. 3B) and no line differences were detected in these egg characteristics (for egg mass: F1,10 = 2.01, p = 0.186 and for yolk mass: F1,10 = 1.25, p = 0.290). Eggshell mass decreased in the second as compared with the first laying cycle (F1,9 = 9.01, p b 0.05; Fig. 3C). Moreover, the HET quail produced eggs with a heavier eggshell when compared with the LET quail (F1,9 = 7.90, p b 0.05) with no interaction between the line and laying cycle (F1,10 = 0.02, p = 0.890).
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Fig. 2. Individual variation of yolk testosterone concentrations between the first and the second laying cycle in the low (A) and high (B) egg testosterone lines of Japanese quail.
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Fig. 3. Egg mass (A), yolk mass (B) and shell mass (C) in the low (LET) and high (HET) egg testosterone lines of Japanese quail in the first and the second laying cycle. Values represent means ± SE.
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4. Discussion We examined the intra- and inter-individual variations of yolk T levels across two laying cycles in the Japanese quail divergently selected for egg T content. To induce the second cycle the quails were forced moulted. Avian moult is a seasonal event and in most free-living birds involves reproductive quiescence followed by restored reproductive activity in the next breeding season. In poultry an induced moult results in the cessation of egg laying and regression of the reproductive system for a limited period (Berry, 2003). Egg production is resumed in the second cycle and generally an improvement of the egg quality is expected (Berry and Brake, 1987; Verheyen and Decuypere, 1991). In quails, we applied a 24-hour-fasting and reduced the photoperiod to 8L:16D for two weeks. This treatment induced moult and stopped egg production in 6 LET and 7 HET females. Afterwards, they were returned to their home cages and after exposure to the stimulatory photoperiod 12 females renewed their egg laying rate to the pre-moult levels 4 weeks later. As expected plasma T concentrations sharply declined under the inhibitory photoperiod. This finding is in accord with the suppressed release of other sex steroid hormones and an involution of the reproductive organs in the forced moulted hens (Hoshino et al., 1988). Decreased plasma T levels in the reproductively quiescent quails probably reflected pituitary gland refractoriness to gonadotropin releasing hormone (GnRH) and a decreased release of gonadotropins as small white follicles within the ovary remain sensitive to exogenous luteinizing hormone (LH) administration (Etches, 1996). Plasma T concentrations did not differ between the HET and LET lines either before or after the treatment. These results correspond with our previous data (Okuliarova et al., 2011a) suggesting that physiological ranges of circulatory T of the mother are not targeted by the selection on yolk T concentrations. Thus, complex changes in the neuroendocrine control including sensitivity of target tissues may be affected by the selection. The line differences in the yolk T levels were stable over the two laying cycles and even more pronounced in the second reproductive season. The consistent line differences correspond with the high repeatability of the yolk androgen concentrations found between two consecutive years in the European starling, Sturnus vulgaris (Eising et al., 2008). On the other hand, in canaries (Serinus canaria) significant intra-female consistency was determined for the amount of yolk T but not yolk T concentrations (Muller et al., 2012). We showed a stability of the line differences between the two laying cycles for both the total content and the concentrations of yolk T indicating that altered steroid biosynthesis and transport mechanisms are more likely to explain the difference between the LET and HET lines than a variation in yolk accumulation. Compared to the pre-moult stage, yolk T concentrations increased in eggs produced in the second laying cycle in the HET females. Such enhancement of the yolk T deposition was not found in the LET quails since more than half of the LET females decreased yolk T levels in their eggs between cycles. Up till now, the inter-seasonal variation of the maternal hormone deposition has not been thoroughly investigated, especially in precocial birds. Previous studies in altricial birds have demonstrated that the maternal deposition of yolk androgens declines between the eggs of the first and second clutches during one breeding season (Schwabl, 1997; Tobler et al., 2007). Similarly, a decline of yolk T levels was found from the early to the latest stages of the first laying cycle in Japanese quails (Okuliarova et al., 2009; Guibert et al., 2012). Moreover, both egg laying intensity and egg quality gradually decrease after the first year of age (Woodard et al., 1973; Joyner et al., 1987) and this damping of reproductive functions is associated with reduced plasma gonadotropin levels and pituitary responsiveness to GnRH in ageing hens (Sharp et al., 1992; Ciccone et al., 2005). A re-exposure to long days following moult restores the pituitary gland responsiveness to GnRH as well as plasma LH concentrations resulting in renewed ovarian functions and sex steroid
production (Hoshino et al., 1988; Sharp et al., 1992). The physiological mechanisms controlling the subsequent recovery of the hypothalamic– pituitary–gonadal axis and egg production are not completely understood yet but the rejuvenation of neuroendocrine regulations and reproductive organs has been proposed (Heryanto et al., 1997; Chowdhury and Yoshimura, 2002). Therefore, an increase of the yolk T deposition in moulted HET but not LET quails may refer to a higher responsiveness of the HET than LET lines to rejuvenation processes either at the central or peripheral level or both. For example, a resumption of laying is accompanied by cell proliferation and apoptosis mediated remodelling of the anterior pituitary (Chowdhury and Yoshimura, 2002) and an increase of the plasma LH associated with increased LH pulse frequency (Bacon and Long, 1996). The differences in yolk T concentrations were not reflected in the plasma T levels, which did not differ between the first and the second cycles in both lines. Since we can expect up-regulated steroidogenesis in the HET females due to their enhanced yolk T deposition, mechanisms that control the concomitant increase of T concentrations in the circulation are also expected. We can predict that females laying eggs with high T levels avoid the undesirable increase of plasma androgen concentrations through enhanced conversion of T into estrogens or biologically inactive metabolites. As the expression of aromatase, catalysing the conversion of T to estradiol, is mainly localised in the theca externa, the outermost layer of the follicular wall (Kato et al., 1995), it is likely that the formed estradiol preferentially enters the circulation. Only low levels pass into the yolk to prevent the interference with the sexual differentiation of the embryo (Adkins-Regan et al., 1995). Further studies are in progress to address these mechanistic issues. The different yolk T depositions of the LET and HET females between the laying cycles were not accompanied by changes of other egg quality characteristics. Both the egg mass and yolk mass did not change between the end of the first and the beginning of the second laying cycle. In chickens, an improvement of eggshell quality has been observed after induced moult although this enhancement is often transient and depends on several factors (Verheyen and Decuypere, 1991; Alodan and Mashaly, 1999). In our study, the eggshell mass decreased in the second laying cycle in both lines. Moreover, the HET quails laid eggs with a heavier eggshell than the LET quails in both laying cycles. This line differences in the eggshell mass were not found in former generations when the line comparison was performed in young 11-week-old quails (Okuliarova et al., 2011a). Eggshell quality is known to decline with increasing age especially as a result of changed calcium metabolism (Joyner et al., 1987). Therefore, our current study suggests that this process could be slower in the HET than LET lines and females laying eggs with high yolk T levels will also produce eggs of better eggshell quality with increasing age as compared with females that have low yolk T deposition. These data are in line with the positive correlation between yolk T concentrations and eggshell mass that was demonstrated in the population of random-bred quails (Okuliarova et al., 2009). We did not evaluate the inter-seasonal variation of other yolk constituents that may change besides yolk T levels. However, in the second generation, the eggs of the HET quails contained higher androstenedione and lower estradiol concentrations than those of the LET quail (Okuliarova et al., 2011a) indicating that the maternal deposition of yolk T correlates with changes of other yolk steroids with potential consequences on offspring development. Embryonic exposure to variable amounts of maternally-derived androgens in the yolk is frequently discussed in an adaptive manner and its consequences on several fitness-related traits of offspring have been widely studied in different avian species (Groothuis et al., 2005). In precocial birds, experimental in ovo injection of T resulted in significant effects on physiology (Cucco et al., 2008) and on the behavioural phenotype of young chicks (Daisley et al., 2005; Okuliarova et al., 2007; Bertin et al., 2008). Moreover, our previous experiments showed that the selection for high egg T content positively affected
M. Zeman et al. / Comparative Biochemistry and Physiology, Part A 164 (2013) 271–275
post-hatching growth (Okuliarova et al., 2011b), whereas elevated growth rate was not compromised by a weaker innate immune response in the HET as compared with the LET quail chicks (Kankova et al., 2012). Therefore, the increase of yolk T concentrations in the second laying cycle in the HET females may reflect an improvement of their breeding value between cycles that together with the better prospects of experienced females to cope with a stable environment can increase their reproductive success. Thus, the inter-seasonal variation in the maternal hormone deposition may be of importance especially for free-living birds with a long lifespan, in which age-specific reproductive success is well described (Wheelwright and Schultz, 1994). In conclusion, the present study demonstrates highly consistent differences in yolk T levels between the LET and HET lines across two subsequent reproductive seasons. The increased yolk T deposition in the second laying cycle in the HET but not the LET quails suggests that the genetic component may contribute to both inter- and intra-female variations in maternal androgens in the egg. Regarding ecological aspects, inter-female differences in yolk T deposition may predict an ability of the mothers to modify their egg hormone content and programme development of their progeny in different reproductive seasons.
Acknowledgements This study was supported by the Slovak Research and Development Agency (APVV 0047-10) and the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and of the Slovak Academy of Sciences (VEGA 1/0686/12).
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Contents lists available at SciVerse ScienceDirect
Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Genetic differences in yolk testosterone levels influence maternal hormone deposition in the second laying cycle in Japanese quails Michal Zeman a, b,⁎, Peter Skrobanek b, Monika Okuliarova a a b
Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic
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Article history: Received 8 August 2012 Received in revised form 20 October 2012 Accepted 20 October 2012 Available online 25 October 2012 Keywords: Genetic variability Induced moult Maternal effects Quail Yolk testosterone
a b s t r a c t 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. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The egg represents the only source of nutritive and biologically active substances that the avian embryo can utilize for its growth and development. Egg composition has been extensively studied in relation to the functional role of essential nutrients such as proteins, lipids, vitamins (Finkler et al., 1998; Royle et al., 1999) and especially the considerable amounts of information carrying compounds transferred into the egg (Schwabl, 1993; Wilson and McNabb, 1997; Grindstaff et al., 2003). Maternal hormones have attracted probably the most attention since they represent a pathway for mediating trans-generational maternal effects. It is thought that the mothers may via hormone deposition into the egg transmit information about the post-hatching environment to the embryo and optimize the development and behaviour of their progeny (Groothuis et al., 2005). The maternal deposition of yolk androgens varies in relation to the environment experienced by the laying female (Gil, 2008) but fluctuations of social and ecological factors themselves are insufficient to explain high inter-female differences in yolk androgen levels documented under stable social and environmental conditions (Okuliarova et al., 2009). Recent studies have proved the genetic component of maternal hormone transfer into the egg on the base of mother–daughter ⁎ Corresponding author at: Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University Bratislava, Mlynska dolina, 842 15 Bratislava, Slovak Republic. Tel.: +421 2 602 96 424. E-mail address: [email protected] (M. Zeman). 1095-6433/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2012.10.028
resemblance in wild populations of the collared flycatcher, Ficedula albicollis (Tschirren et al., 2009) and a long-term selection experiment in the precocial Japanese quail, Coturnix japonica (Okuliarova et al., 2011a). The heritable variation of yolk androgen concentrations can account for nearly half of a total phenotypic variance of this trait (Okuliarova et al., 2011a) and it is essential for an understanding of evolutionary consequences of hormone-mediated maternal effects (Mousseau and Fox, 1998). Besides the high inter-individual variability in maternal hormone deposition marked intra-individual differences have been demonstrated in several avian species (Groothuis et al., 2005). In particular, altricial birds can produce a distinct pattern of yolk androgen concentrations within a single clutch (Schwabl, 1993; Eising et al., 2001) as well as between successively laid clutches (Schwabl, 1997; Gil et al., 2006; Tobler et al., 2007). This between-clutch pattern of yolk androgen deposition was not investigated in free-living precocial birds yet but a decline of yolk testosterone (T) concentrations was found between the early and the latest stages of the first reproductive cycle in domestic species (Okuliarova et al., 2011a; Guibert et al., 2012). Regarding this intra-individual variability, limited studies deal with changes of maternal hormone deposition across the longer period of the reproductive lifespan and in association with age-specific reproductive success that can be important from both the evolutionary and ecological perspectives. In domestic fowls, the duration of the egg producing period is considerably extended although both the egg laying intensity and egg quality gradually decrease after the first year of age (Woodard et al.,
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1973; Joyner et al., 1987). To restore reproductive performance to its former levels, egg producers induce moulting in their flocks applying short-term starvation and reducing the photoperiod. Such induced moulting results in the cessation of egg laying and reproductive quiescence for a limited period (a couple of weeks), which is followed by the second reproductive cycle (Berry, 2003). In the present study, we investigated whether maternal hormone deposition into the eggs varies between the first and the second laying cycles in precocial Japanese quail. We used genetic lines divergently selected for high (HET line) and low (LET line) egg T contents that can provide a useful model to study sources of yolk androgen variability represented by both the genetic and environmental component and genotype–environment interactions. To address how the genetically determined differences in maternal hormone deposition relate to female's breeding quality across two laying cycles we measured yolk and plasma T concentrations and egg quality traits in the HET and LET females at the end of the first and at the beginning of the second laying cycle after forced moulting. 2. Material and methods 2.1. Animals and moulting procedure Adult Japanese quail (C. japonica) from the fourth generation of the LET and HET lines divergently selected for yolk T concentrations (Okuliarova et al., 2011a) were used in the study. At the age of 46 weeks 16 pairs (8 LET and 8 HET) were taken from the original breeding population. Each female was housed with a familiar male from the same line in an individual cage (0.22 m 2). These pairs were kept stable for at least 4 weeks before starting the experiment. All birds were maintained in the same room at controlled temperatures (22 ± 2 °C) under a stimulatory photoperiod of 14 h of light (L) and 10 h of dark (D) with food and water ad libitum. For 2 weeks, daily egg production was recorded and the mean ± standard error of mean (SE) laying rates were 93.3 ± 4.7% and 85.6 ± 3.4% for the LET and HET females, respectively. At the end of this period the last 2 eggs per female were collected for yolk T analysis. A standard moulting procedure was applied to stop the laying and induce the second cycle (Berry, 2003). Females were placed into group cages according to the line and exposed to an inhibitory photoperiod of 8L:16D for 14 consecutive days. Food was removed for the first 24 h and for the rest of the treatment food and water were provided ad libitum. During the short days 6 LET and 7 HET females stopped laying and were moulting heavily. First, the feathers on both sides of the breast were lost, followed by secondary and primary reminges. The quails were then returned to their home cages with their males and re-exposed to the stimulatory photoperiod of 14L:10D. Twelve females (6 LET and 6 HET) restored regular egg production within 4 weeks and only eggs from those females were used for statistical analyses. Four weeks after the re-exposure to the stimulatory photoperiod, the eggs were collected for analyses across 6 days when the mean (±SE) laying rate was the same for both lines (83.3± 4.4%). The care and use of animals was in accordance with laws and regulations of the Slovak Republic and approved by the Ethical Committee of the Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic.
albumen, were thoroughly homogenized and stored at − 20 °C until steroid extraction and yolk T assay. Blood samples were obtained from the wing vein three times: 1) during the first laying cycle (39 weeks of age), 2) during the moulting (11 days after the shortening the photoperiod) and 3) during the second laying cycle (5 weeks after the re-exposure to the stimulatory photoperiod). Blood was collected in the morning, about 6–8 h before a predicted oviposition. Plasma was stored at −20 °C until T analysis. 2.3. Testosterone assay Testosterone was extracted from the yolk using the procedure described in detail by Okuliarova et al. (2009). Average recovery was 63.7± 0.7%. Testosterone concentrations were measured in both yolk extracts (20 μL aliquot) and plasma samples (50 μL aliquot) by radioimmunoassay using [1,2,6,7-3H]-T (Amersham Biosciences, UK, specific activity 3.52 TBq/mmol) and a specific antibody generated in rabbits against testosterone-3-(carboxy-methyl) oxime bovine serum albumin conjugate. The cross-reactivity of the antiserum was 9.6% with 5α-dihydrotestosterone, 0.1% with androstenediol and lower than 0.1% with other steroids (Zeman et al., 1986). Yolk and plasma samples were run separately within 4 assays with a mean intra-assay variation coefficient of 4% and inter-assay variation coefficient of 5.2%. The mean (±SE) assay sensitivity was 2.16 ± 0.16 pg/tube. 2.4. Statistical analyses The data for the yolk T levels and egg characteristics were averaged per individual female per laying cycle and analysed using repeated-
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2.2. Eggs and blood samples In total we collected 46 and 43 eggs from the LET and HET quails, respectively. Two eggs per female were analysed in the first laying cycle and 5.4 ± 0.2 (mean ± SE) eggs per female at the beginning of the second cycle. First, we measured the egg quality characteristics: egg mass, yolk mass and eggshell mass. The eggshell was weighed after cleaning and drying at 80 °C. The yolks, separated from the
Fig. 1. Plasma (A) and egg yolk (B) testosterone concentrations in the low (LET, N = 6) and high (HET, N=6) egg testosterone lines of Japanese quail in the first and the second laying cycle. Values represent means±SE. Different letters above the bars denote significant differences between groups (pb 0.05).
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measures analysis of variance with line as the between-group factor, laying cycle as the within-group factor and their interaction. Yolk T concentrations and total yolk T content were log transformed to fit the normal distribution and homogeneity of variance. All other data met these assumptions. Differences between groups were evaluated using Fisher's LSD post hoc tests. 3. Results The T concentrations in the female's plasma were not influenced by line (F1,10 = 0.14, p = 0.713), laying cycle (F1,10 = 1.09, p = 0.322) or the interaction between both factors (F1,10 = 0.70, p = 0.422) (Fig. 1A). During the period of forced moulting plasma T levels declined under the detection limit in all females (lower than 35 pg/mL) except for one that stopped laying eggs a few days after the blood sample collection. As expected mean yolk T concentrations in the egg were higher in the HET compared with the LET line (F1,10 = 29.14, p b 0.001). The effect of the laying cycle on yolk T concentrations was not significant (F1,10 = 2.47, p = 0.147) but a significant interaction between the line and laying cycle was found (F1,10 = 14.64, p b 0.01). Post hoc comparison revealed that yolk T concentrations had increased in eggs of the second laying cycle when compared with eggs of the first cycle in the HET quail (p b 0.01) while no significant change was found in the LET quail (Fig. 1B). Looking at the inter-female variation the majority of the LET quails showed a numerical decrease of yolk T deposition across two laying cycles (Fig. 2A). In contrast, all females except one in the HET line had increased yolk T levels in their eggs in the second laying cycle (Fig. 2B). The same pattern as for yolk T concentrations was found for total T content in the yolk (line: F1,10 = 36.21, p b 0.001; laying cycle:
F1,10 = 1.21, p = 0.298 and the interaction of line× laying cycle: F1,10 = 12.99, p b 0.01). The mean T levels (±SE) for the whole yolk were 22.1± 1.7 ng and 41.1± 3.7 ng at the end of the first cycle and 18.7 ± 2.1 ng and 60.8 ±10.2 ng at the beginning of the second cycle in the LET and HET lines, respectively. The second laying cycle did not affect either egg mass (F1,10 = 0.01, p = 0.935; Fig. 3A) or yolk mass (F1,10 = 0.11, p = 0.752; Fig. 3B) and no line differences were detected in these egg characteristics (for egg mass: F1,10 = 2.01, p = 0.186 and for yolk mass: F1,10 = 1.25, p = 0.290). Eggshell mass decreased in the second as compared with the first laying cycle (F1,9 = 9.01, p b 0.05; Fig. 3C). Moreover, the HET quail produced eggs with a heavier eggshell when compared with the LET quail (F1,9 = 7.90, p b 0.05) with no interaction between the line and laying cycle (F1,10 = 0.02, p = 0.890).
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Fig. 2. Individual variation of yolk testosterone concentrations between the first and the second laying cycle in the low (A) and high (B) egg testosterone lines of Japanese quail.
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Fig. 3. Egg mass (A), yolk mass (B) and shell mass (C) in the low (LET) and high (HET) egg testosterone lines of Japanese quail in the first and the second laying cycle. Values represent means ± SE.
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4. Discussion We examined the intra- and inter-individual variations of yolk T levels across two laying cycles in the Japanese quail divergently selected for egg T content. To induce the second cycle the quails were forced moulted. Avian moult is a seasonal event and in most free-living birds involves reproductive quiescence followed by restored reproductive activity in the next breeding season. In poultry an induced moult results in the cessation of egg laying and regression of the reproductive system for a limited period (Berry, 2003). Egg production is resumed in the second cycle and generally an improvement of the egg quality is expected (Berry and Brake, 1987; Verheyen and Decuypere, 1991). In quails, we applied a 24-hour-fasting and reduced the photoperiod to 8L:16D for two weeks. This treatment induced moult and stopped egg production in 6 LET and 7 HET females. Afterwards, they were returned to their home cages and after exposure to the stimulatory photoperiod 12 females renewed their egg laying rate to the pre-moult levels 4 weeks later. As expected plasma T concentrations sharply declined under the inhibitory photoperiod. This finding is in accord with the suppressed release of other sex steroid hormones and an involution of the reproductive organs in the forced moulted hens (Hoshino et al., 1988). Decreased plasma T levels in the reproductively quiescent quails probably reflected pituitary gland refractoriness to gonadotropin releasing hormone (GnRH) and a decreased release of gonadotropins as small white follicles within the ovary remain sensitive to exogenous luteinizing hormone (LH) administration (Etches, 1996). Plasma T concentrations did not differ between the HET and LET lines either before or after the treatment. These results correspond with our previous data (Okuliarova et al., 2011a) suggesting that physiological ranges of circulatory T of the mother are not targeted by the selection on yolk T concentrations. Thus, complex changes in the neuroendocrine control including sensitivity of target tissues may be affected by the selection. The line differences in the yolk T levels were stable over the two laying cycles and even more pronounced in the second reproductive season. The consistent line differences correspond with the high repeatability of the yolk androgen concentrations found between two consecutive years in the European starling, Sturnus vulgaris (Eising et al., 2008). On the other hand, in canaries (Serinus canaria) significant intra-female consistency was determined for the amount of yolk T but not yolk T concentrations (Muller et al., 2012). We showed a stability of the line differences between the two laying cycles for both the total content and the concentrations of yolk T indicating that altered steroid biosynthesis and transport mechanisms are more likely to explain the difference between the LET and HET lines than a variation in yolk accumulation. Compared to the pre-moult stage, yolk T concentrations increased in eggs produced in the second laying cycle in the HET females. Such enhancement of the yolk T deposition was not found in the LET quails since more than half of the LET females decreased yolk T levels in their eggs between cycles. Up till now, the inter-seasonal variation of the maternal hormone deposition has not been thoroughly investigated, especially in precocial birds. Previous studies in altricial birds have demonstrated that the maternal deposition of yolk androgens declines between the eggs of the first and second clutches during one breeding season (Schwabl, 1997; Tobler et al., 2007). Similarly, a decline of yolk T levels was found from the early to the latest stages of the first laying cycle in Japanese quails (Okuliarova et al., 2009; Guibert et al., 2012). Moreover, both egg laying intensity and egg quality gradually decrease after the first year of age (Woodard et al., 1973; Joyner et al., 1987) and this damping of reproductive functions is associated with reduced plasma gonadotropin levels and pituitary responsiveness to GnRH in ageing hens (Sharp et al., 1992; Ciccone et al., 2005). A re-exposure to long days following moult restores the pituitary gland responsiveness to GnRH as well as plasma LH concentrations resulting in renewed ovarian functions and sex steroid
production (Hoshino et al., 1988; Sharp et al., 1992). The physiological mechanisms controlling the subsequent recovery of the hypothalamic– pituitary–gonadal axis and egg production are not completely understood yet but the rejuvenation of neuroendocrine regulations and reproductive organs has been proposed (Heryanto et al., 1997; Chowdhury and Yoshimura, 2002). Therefore, an increase of the yolk T deposition in moulted HET but not LET quails may refer to a higher responsiveness of the HET than LET lines to rejuvenation processes either at the central or peripheral level or both. For example, a resumption of laying is accompanied by cell proliferation and apoptosis mediated remodelling of the anterior pituitary (Chowdhury and Yoshimura, 2002) and an increase of the plasma LH associated with increased LH pulse frequency (Bacon and Long, 1996). The differences in yolk T concentrations were not reflected in the plasma T levels, which did not differ between the first and the second cycles in both lines. Since we can expect up-regulated steroidogenesis in the HET females due to their enhanced yolk T deposition, mechanisms that control the concomitant increase of T concentrations in the circulation are also expected. We can predict that females laying eggs with high T levels avoid the undesirable increase of plasma androgen concentrations through enhanced conversion of T into estrogens or biologically inactive metabolites. As the expression of aromatase, catalysing the conversion of T to estradiol, is mainly localised in the theca externa, the outermost layer of the follicular wall (Kato et al., 1995), it is likely that the formed estradiol preferentially enters the circulation. Only low levels pass into the yolk to prevent the interference with the sexual differentiation of the embryo (Adkins-Regan et al., 1995). Further studies are in progress to address these mechanistic issues. The different yolk T depositions of the LET and HET females between the laying cycles were not accompanied by changes of other egg quality characteristics. Both the egg mass and yolk mass did not change between the end of the first and the beginning of the second laying cycle. In chickens, an improvement of eggshell quality has been observed after induced moult although this enhancement is often transient and depends on several factors (Verheyen and Decuypere, 1991; Alodan and Mashaly, 1999). In our study, the eggshell mass decreased in the second laying cycle in both lines. Moreover, the HET quails laid eggs with a heavier eggshell than the LET quails in both laying cycles. This line differences in the eggshell mass were not found in former generations when the line comparison was performed in young 11-week-old quails (Okuliarova et al., 2011a). Eggshell quality is known to decline with increasing age especially as a result of changed calcium metabolism (Joyner et al., 1987). Therefore, our current study suggests that this process could be slower in the HET than LET lines and females laying eggs with high yolk T levels will also produce eggs of better eggshell quality with increasing age as compared with females that have low yolk T deposition. These data are in line with the positive correlation between yolk T concentrations and eggshell mass that was demonstrated in the population of random-bred quails (Okuliarova et al., 2009). We did not evaluate the inter-seasonal variation of other yolk constituents that may change besides yolk T levels. However, in the second generation, the eggs of the HET quails contained higher androstenedione and lower estradiol concentrations than those of the LET quail (Okuliarova et al., 2011a) indicating that the maternal deposition of yolk T correlates with changes of other yolk steroids with potential consequences on offspring development. Embryonic exposure to variable amounts of maternally-derived androgens in the yolk is frequently discussed in an adaptive manner and its consequences on several fitness-related traits of offspring have been widely studied in different avian species (Groothuis et al., 2005). In precocial birds, experimental in ovo injection of T resulted in significant effects on physiology (Cucco et al., 2008) and on the behavioural phenotype of young chicks (Daisley et al., 2005; Okuliarova et al., 2007; Bertin et al., 2008). Moreover, our previous experiments showed that the selection for high egg T content positively affected
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post-hatching growth (Okuliarova et al., 2011b), whereas elevated growth rate was not compromised by a weaker innate immune response in the HET as compared with the LET quail chicks (Kankova et al., 2012). Therefore, the increase of yolk T concentrations in the second laying cycle in the HET females may reflect an improvement of their breeding value between cycles that together with the better prospects of experienced females to cope with a stable environment can increase their reproductive success. Thus, the inter-seasonal variation in the maternal hormone deposition may be of importance especially for free-living birds with a long lifespan, in which age-specific reproductive success is well described (Wheelwright and Schultz, 1994). In conclusion, the present study demonstrates highly consistent differences in yolk T levels between the LET and HET lines across two subsequent reproductive seasons. The increased yolk T deposition in the second laying cycle in the HET but not the LET quails suggests that the genetic component may contribute to both inter- and intra-female variations in maternal androgens in the egg. Regarding ecological aspects, inter-female differences in yolk T deposition may predict an ability of the mothers to modify their egg hormone content and programme development of their progeny in different reproductive seasons.
Acknowledgements This study was supported by the Slovak Research and Development Agency (APVV 0047-10) and the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and of the Slovak Academy of Sciences (VEGA 1/0686/12).
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