University of Groningen In ovo testosterone treatment reduces long-term survival of female pigeons Matson, K. D.; Riedstra, B.; Tieleman, B. I. Published in: Journal of Animal Physiology and Animal Nutrition DOI: 10.1111/jpn.12469 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2016 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Matson, K. D., Riedstra, B., & Tieleman, B. I. (2016). In ovo testosterone treatment reduces long-term survival of female pigeons: a preliminary analysis after nine years of monitoring. 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For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 06-01-2024 DOI: 10.1111/jpn.12469 ORIGINAL ARTICLE In ovo testosterone treatment reduces long-term survival of female pigeons: a preliminary analysis after nine years of monitoring K. D. Matson1, B. Riedstra2 and B. I. Tieleman2 1 Resource Ecology Group, Department of Environmental Sciences, Wageningen University, Wageningen, The Netherlands, and 2 Groningen Institute for Evolutionary Life Sciences, University of Groningen, Groningen, The Netherlands Summary Early exposure to steroid hormones, as in the case of an avian embryo exposed yolk testosterone, can impact the biology of an individual in different ways over the course of its life. While many early-life effects of yolk testosterone have been documented, later-life effects remain poorly studied. We followed a cohort of twenty captive pigeons hatched in 2005. Half of these birds came from eggs with experimentally increased concentrations of testosterone; half came from control eggs. Preliminary results suggest non-random mortality during the birds’ first nine years of life. Hitherto, all males have survived, and control females have survived better than testosterone-treated ones. Despite inherent challenges, studies of later-life consequences of early-life exposure in longer-lived species can offer new perspectives that are precluded by studies of immediate outcomes or shorterlived species. Keywords ageing, bird, egg, hormone, maternal effect, mortality Correspondence Kevin D. Matson, Resource Ecology Group, Department of Environmental Sciences, Wageningen University, P.O. Box 47, 6700 AA Wageningen, The Netherlands. Tel: +31 317481782; E-mail: kevin.matson@wur.nl Received: 30 September 2015; accepted: 9 December 2015 Introduction Exposure to steroid hormones impacts the biology of animals in many ways. The specific nature of these effects depends on, among other factors, timing. For instance, both the ontogenetic stage at exposure and the time elapsed since exposure can shape a hormone’s influence (Eising et al., 2006; Gil, 2008; Weinstock, 2008; Tobler et al., 2009; Riedstra et al., 2013). One type of pertinent early-life experience is prenatal (in utero or in ovo) exposure of offspring to maternal hormones (Gil, 2008; Weinstock, 2008). Indeed, female birds deposit steroid hormones in the yolks of their eggs, and much research on this phenomenon and its consequences has focused on testosterone, an androgen whose presence in yolk was first reported by Schwabl (1993). Maternal yolk testosterone has benefits and costs for offspring. These benefits and costs can manifest themselves at different points in a bird’s lifetime, yet most research focuses on early life (Sockman and Schwabl, 2000; Eising et al., 2006; Gil, 2008; Groothuis and Schwabl, 2008). Variability in naturally deposited concentrations of yolk testosterone is thought to serve as a mechanism for developmental plasticity in offspring, so that offspring are as prepared as possible for the post-hatch conditions they face (Gil, 2008). The nature of those preparations, however, depends on study species, sex and various experimental conditions. During the post-hatch period, nestling behaviour (e.g. begging), physiology (e.g. metabolism), immunology (e.g. antibody production), morphology (e.g. body size and colour) and other domains can be influenced by prenatal testosterone exposure (Sockman and Schwabl, 2000; Pilz et al., 2004; Gil, 2008; Tobler et al., 2009; Riedstra et al., 2013). While the effects of absolute level and variability of testosterone concentration on these domains are often considered within a framework of Darwinian fitness (Eising et al., 2006; Groothuis and Schwabl, 2008), effects on moreintegrative proxies, for example nestling survival (Sockman and Schwabl, 2000; Pilz et al., 2004), are also sometimes reported. On balance, young birds might gain a survival advantage from increased testosterone, at least during the phase of parental dependency, but the broader biological context still matters. Journal of Animal Physiology and Animal Nutrition 100 (2016) 1031–1036 © 2016 Blackwell Verlag GmbH 1031 In ovo testosterone reduces long-term survival of pigeons As Gil (2008) concludes, more testosterone is probably not always better, and costs borne by adults are especially unclear. Later-life repercussions of yolk testosterone are underexplored component of the cost–benefit balance pertaining to testosterone’s effects on fitness (Gil, 2008). A few studies examine the relationship between egg androgens and recruitment into local breeding populations, but the results are mixed (Tschirren et al., 2007; Hegyi et al., 2011; Ruuskanen et al., 2012). Other studies that include measurements beyond the period of post-natal development and into the period of sexual maturity in males and females generally relate to the long-lasting organizing effects of testosterone on behaviour, physiology, immunology and secondary sexual differentiation (Gil, 2008; Groothuis and Schwabl, 2008; Goerlich et al., 2009; Riedstra et al., 2013). For example, Tobler et al. (2009) show that experimentally increasing testosterone concentration of eggs leads to stronger primary and secondary antibody responses in five- and seven-month-old Zebra finches (Taeniopygia guttata), and Eising et al. (2006) show that a combined androgen treatment (testosterone and androstenedione) of eggs leads to more-frequent sexual and aggressive behaviours and more adult-like plumage in ten-month-old Black-headed gulls (Larus ridibundus). Both early- and later-life effects of yolk testosterone can be sex specific (Adkins-Regan et al., 2013). Under natural conditions, such sex specificity is obvious: testosterone is predominantly a masculinizing mechanism and a driver of male biology (although during avian development, estrogens also play a role in masculinization, e.g. Rochester et al., 2008). Yet in experimental studies, interactions between sex and testosterone treatment are not always apparent. For instance, in both examples cited above (Eising et al., 2006; Tobler et al., 2009), the investigators tested for and failed to find significant interactions of this type. One long-term study that does show sex-specific effects of in ovo testosterone treatment focuses on agespecific mortality in house sparrows (Passer domesticus) over a 3.5-year period (Schwabl et al., 2011). This study, which to the best of our knowledge is the only study to date linking yolk testosterone and adult survival, suggests that elevated testosterone is protective (i.e. reduces the mortality hazard) and that females benefit more than males from this protective effect. Given the dearth of long-term survival data, we report the effects of in ovo testosterone treatment on male and female survival until nine years of age in a small cohort of homing pigeons (Columba livia domestica). Pigeons become sexually mature by approximately 1032 K. D. Matson, B. Riedstra and B. I. Tieleman six months of age, raise multiple two-egg clutches per year and have a maximum lifespan potential (MLSP) of 35 years (Montgomery et al., 2011; Human Ageing Genomic Resources, 2015). Materials and methods We studied twenty homing pigeons (twelve ♀, eight ♂) hatched late in 2005 (Table 1). Details about the care and use of these birds have been published previously (van de Crommenacker et al., 2010), so we only summarize key information here. Single-sex groups of four individuals were housed separately in five covered, open-air aviaries. All birds were exposed to outside air temperature and natural photoperiod and were provided with ad libitum food and water. Our work with these animals complied with all applicable institutional regulations (University of Groningen Animal Experimentation Committee, license no. 5095) and Dutch and European laws. Two experimental manipulations involved treating only half of each sex (Table 1). The remaining birds served as controls. Despite being nearly three years apart, the two manipulations were balanced within each sex and housing group. First, in 2005 we injected freshly laid eggs either with 16 ng of testosterone in 0.1 ml sesame oil or with 0.1 ml sesame oil only. The testosterone injection, designed as a uniform procedure to increase concentrations of the hormone in both first and second eggs in the light of the overall physiological range, was based on the data available in 2005 (relevant data from concurrent experiments are available in Goerlich et al., 2009). Two eggs within a clutch always received the identical injections, and all clutches were produced by unmanipulated parental pairs. The twenty individuals in this study hatched from eggs that were first, second or from an unknown position within their clutch. At the time of the second manipulation in August 2008, the birds were adults. On six consecutive days, we orally dosed individuals either with 180 mg of lysozyme (L6876; Sigma, St. Louis, MO, USA) in 1 ml phosphate-buffered saline (PBS, P4417; Sigma) or with 1 ml of PBS only (van de Crommenacker et al., 2010). All other procedures were applied consistently to all birds. These universal procedures included the following: weighing, behavioural testing, blood sampling, cloacal and choanal swabbing, fasting, temporary isolation for quantifying food consumption and basal metabolic rate, and de-worming. In 2006, all birds received an injection of 0.5 ml of a 2% suspension of sheep red blood cells. In 2008, all birds received an injection of 2 ml/kg body mass of a 1.25 mg/ml solu- Journal of Animal Physiology and Animal Nutrition 100 (2016) 1031–1036 © 2016 Blackwell Verlag GmbH In ovo testosterone reduces long-term survival of pigeons K. D. Matson, B. Riedstra and B. I. Tieleman Table 1 Traits of the twenty individual homing pigeons in the study Bird No. Lay Order Hatch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 ? 1 2 1 ? 1 2 1 ? 1 1 2 1 ? 1 2 1 1 1 25 Dec. 2005 30 Nov. 2005 22 Dec. 2005 24 Dec. 2005 23 Dec. 2005 22 Dec. 2005 25 Dec. 2005 24 Dec. 2005 23 Dec. 2005 24 Dec. 2005 24 Dec. 2005 23 Dec. 2005 26 Dec. 2005 23 Dec. 2005 20 Dec. 2005 26 Dec. 2005 26 Dec. 2005 24 Dec. 2005 22 Dec. 2005 22 Dec. 2005 Death Age at Death (year) Cause Sex Testo* SRBC† Lyso‡ LPS§ Group¶ 6 Feb. 2011 2 April 2013 17 July 2013 29 Jan. 2011 28 Sept. 2012 – 17 July 2013 – – – – – – – – – – – – – 5.1 7.3 7.6 5.1 6.8 – 7.6 – – – – – – – – – – – – – Euthanized Died Died Died Euthanized – Euthanized – – – – – – – – – – – – – F F F F F F F F F F F F M M M M M M M M Yes Yes Yes Yes Yes Yes No No No No No No Yes Yes Yes Yes No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No Yes Yes Yes No No No Yes Yes No No Yes Yes No No Yes Yes** Yes Yes Yes Yes** Yes Yes Yes** Yes Yes Yes** Yes Yes Yes Yes Yes Yes Yes Yes 1 5 2 1 2 5 1 2 5 1 2 5 3 4 3 4 3 4 3 4 Notes Clutch mate of no. 5 Clutch mate of no. 4 Clutch mate of no. 9 Clutch mate of no. 8 *Testosterone, in ovo in 2005. †Sheep red blood cells, injection in 2006. ‡Lysozyme, orally in August 2008. §Lipopolysaccharide, injection in August 2008 or before. **Two injections. ¶Initial housing groups maintained until three of four groupmates died (only group 1 on 17 July 13); then groups 1 and 2 were merged to form a new three member group. tion of lipopolysaccharide (LPS; L7261; Sigma) dissolved in PBS (van de Crommenacker et al., 2010). A subset (four ♀) had already received an earlier LPS injection in a pilot study. We used contingency tables and exact tests to evaluate associations between characteristics of the birds (i.e. male/female, control/treatment) and their survival until nine years of age (i.e. alive/dead). Results By 1 January 2015, the 14 surviving pigeons were all just over nine years of age (Table 1). Six birds, all female, had died (median lifespan: 7.1 years, range: 5.1–7.6 years). The four sex by testosterone treatment groups (control males, testosterone-treated males, control females, testosterone-treated females) differed in survival (Fisher–Freeman–Halton test of 4 9 2 table, n = 20, p = 0.011; if either member of each of the two same-sex sibling pairs is discarded, n = 18, p = 0.024; Freeman and Halton, 1951). Analogous analyses revealed no significant effects of housing group (i.e. aviary) or of lysozyme, the only other experimental treatment not experienced by all twenty pigeons. While all males survived, control females survived better than testosterone-treated ones (Fisher–Boschloo test of 2 9 2 table, test statistic = 0.080, p = 0.039, power = 0.68; Lydersen et al., 2009). To check the robustness of these results, we repeated the analyses using Kaplan–Meier curve comparisons (either the four sex by testosterone treatment groups or only control and testosterone-treated females, Fig. 1) and Cox proportional hazards regression analysis (only control and testosterone-treated females, as males were invariable); all gave similarly significant results (all p < 0.05). Discussion With their MLSP of 35 years, pigeons have served as a model of longevity in studies of the physiology of ageing (Montgomery et al., 2011; Human Ageing Genomic Resources, 2015). Using such a model to study the effects of early-life exposure to testosterone on mortality, the ultimate late-life consequence and an Journal of Animal Physiology and Animal Nutrition 100 (2016) 1031–1036 © 2016 Blackwell Verlag GmbH 1033 In ovo testosterone reduces long-term survival of pigeons K. D. Matson, B. Riedstra and B. I. Tieleman Fig. 1 Survival of female pigeons hatched from control and testosterone-treated eggs over the nine-year study period. Males, none of which have died, are not shown. important determinant of lifetime reproductive success in long-lived species (Clutton-Brock, 1988), not only offers new perspectives (Groothuis and Schwabl, 2008) but also presents notable challenges. At the nine-year mark, the in ovo testosterone treatment had exerted sex-specific effects on survival. With testosterone-treated females dying before control females, in ovo testosterone treatment could limit lifetime reproductive success. As in the study of house sparrows by Schwabl et al. (2011), the precise mechanisms mediating the effects of testosterone on survival were impossible to pin down in the current study (but for discussions of potential mechanisms, see Gil, 2008; Groothuis and Schwabl, 2008; Schwabl et al., 2011). Our pigeons were never allowed to reproduce (sexes housed separately), but females routinely laid eggs during most months in all years. This one-sided investment in (attempted) reproduction could contribute to the sex specificity of our results. Additionally, while our pigeons were isolated from predators, the same cannot be said about parasites (pers. obs. of lice, ticks, intestinal worms, haematozoa) or microbes more generally (Matson et al., 2015). In the light of the long-lasting organizing effects of yolk testosterone on the immune system (Glick and Sadler, 1961; Gil, 2008; Tobler et al., 2009), control and testosterone-treated females might have responded differently to similar immunological triggers (Schwabl et al., 2011). 1034 The apparent sex-specific testosterone effect discussed above could disappear in the future if most or all testosterone-treated males die before the control males. But this scenario would almost certainly result in an overall negative effect of testosterone on survival. Furthermore, with only one treated female still living, the effect of testosterone within females is nearly fixed. (In fact, of the 720 possible permutations of deaths of the remaining six females, only one outcome, that is if the sole surviving testosterone-treated female outlives all five remaining control females, would eliminate the significant effect of testosterone on female survival.) While yolk testosterone treatment negatively impacted survival until nine years of age in female pigeons, a similar treatment was previously found to be protective in female house sparrows (Schwabl et al., 2011). We can only speculate about the causes of this discrepancy. As might be expected for two species from different taxonomic orders, many lifehistory traits differ between pigeons and house sparrows (Human Ageing Genomic Resources, 2015). Pigeons have a greater MLSP than house sparrows (23 years), and other longevity data in these species paint a similar picture (Montgomery et al., 2011; Schwabl et al., 2011; Human Ageing Genomic Resources, 2015). But rather than a single trait like longevity, a species’ relative position on a multivariate ‘slow–fast’ life-history continuum (Ricklefs, 2000) might govern Journal of Animal Physiology and Animal Nutrition 100 (2016) 1031–1036 © 2016 Blackwell Verlag GmbH In ovo testosterone reduces long-term survival of pigeons K. D. Matson, B. Riedstra and B. I. Tieleman the costs and benefits of yolk testosterone experienced by males and females in different age classes. Regardless, in combination, our current results and the contradictory results of Schwabl et al. (2011) allow us to recapitulate and extend previous conclusions: these studies of adult survival show that broader biological context, including study species identity, matters and that more testosterone is not always better, just as with early-life consequences. Practical considerations related to maintaining and individually monitoring pigeons or other long-lived animals for years or decades (e.g. space, costs) have likely contributed to the focus on short-term studies of early-life consequences (Gil, 2008). These same considerations tend to constrain sample sizes, but in the current study with six females per group, the testosterone effect was clear. Of the six dead pigeons, three were found dead from unknown causes. The other three were found sufficiently close to death that animal caretakers, who were blind to experimental treatments and research questions, were compelled by animal welfare regulations to euthanize the birds to end their suffering. No decision to euthanize resulted from an isolated traumatic injury (e.g. a broken wing) but instead from more general indicators (e.g. extreme weight loss, failure to move when approached). Notably, the last bird euthanized was the first death of a control bird, thereby weakening the statistical results References Adkins-Regan, E.; Banerjee, S. B.; Correa, S. M.; Schweitzer, C., 2013: Maternal effects in quail and zebra finches: behavior and hormones. General and Comparative Endocrinology 190, 34–41. Clutton-Brock, T. H., 1988: Reproductive Success: Studies of Individual Variation in Contrasting Breeding Systems. University of Chicago Press, Chicago, IL, USA. van de Crommenacker, J.; Horrocks, N. P. C.; Versteegh, M. A.; Komdeur, J.; Tieleman, B. 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