Post-natal exposure to corticosterone affects standard metabolic rate in the zebra finch (Taeniopygia guttata) Spencer, K. A.

University of Groningen Post-natal exposure to corticosterone affects standard metabolic rate in the zebra finch (Taeniopygia guttata) Spencer, K. A.; Verhulst, Simon Published in: General and Comparative Endocrinology DOI: 10.1016/j.ygcen.2008.09.007 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: 2008 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Spencer, K. A., & Verhulst, S. (2008). Post-natal exposure to corticosterone affects standard metabolic rate in the zebra finch (Taeniopygia guttata). General and Comparative Endocrinology, 159(2-3), 250-256. DOI: 10.1016/j.ygcen.2008.09.007 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 11-02-2018

General and Comparative Endocrinology 159 (2008) 250 256 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen Post-natal exposure to corticosterone affects standard metabolic rate in the zebra finch (Taeniopygia guttata) K.A. Spencer a, *, S. Verhulst b a Divi sion of Envi ron men tal and Evo lu tion ary Biol ogy, Gra ham Kerr Build ing, Uni ver sity of Glas gow, Glas gow G12 8QQ, UK b Behav ioural Biol ogy, Ri jksuniversiteit Gron in gen, Ha ren, The Neth er lands article info Article history: Received 15 August 2007 Revised 27 August 2008 Accepted 16 September 2008 Available online 26 September 2008 Key words: Metab o lism Sleep Arousal Stress hor mones Behav ior Met a bolic reg u la tion abstract Post-natal stress has been shown to have impor tant short and long term effects on many adult traits in birds. Dur ing stress, met a bolic alter a tions often result in the mobi li za tion of energy away from energysen si tive func tions such as growth, which could have sig nifi cant impli ca tions for devel op ing ani mals. How ever, little is known about the impli ca tions of stress hor mones for energy con sump tion in grow ing indi vid u als. We exper i men tally increased cor ti co ste rone (CORT) lev els in nest ling zebra finches via oral admin is tra tion, between the ages of 7 and 18 days. The stan dard met a bolic rate (SMR) of birds was measured twice over night when birds were between 11 13 and 55 65 days of age. Devel op men tal CORT admin is tra tion sig nifi cantly ele vated over night var i abil ity in SMR (sd) in nest ling birds (dur ing the treatment period), but not at 55 65 days (5 6 weeks after the treat ment period). The effect on var i abil ity was seen more prom i nently in birds from larger brood sizes and in females. We found no effects of our treat ments on mean SMR over night. How ever, brood size and sex had inter ac tive effects, with males from larger brood sizes hav ing higher SMR at 55 65 days of age. These results sug gest that stress hor mones can have sig nifi cant effects on energy metab o lism and pos si bly noc tur nal arousal and sleep frag men ta tion. How ever, there were no detect able long term effects of our treat ments on SMR, sug gest ing that these effects are only short-lived, in order to main tain homeo sta sis in the short term. 2008 Else vier Inc. All rights reserved. 1. Intro duc tion Stress ful con di tions, such as reduced food avail abil ity, promote the release of adre nal glu co cor ti coid hor mones (Wing field, 1994, 1998), the main such hor mone in birds being cor ti co sterone. Whilst short term increases in cor ti co ste rone are adap tive, per sis tent high lev els result ing from con tin ued stress can have pronounced del e te ri ous effects (Wing field, 1994, 1998). There is consid er able evi dence that ele vated stress hor mone lev els can have sig nifi cant effects on energy metab o lism in adults of a wide range of ani mals (Ast hei mer et al., 1992; De Bo eck et al., 2001; Laug ero and Mo berg, 2000a,b; Wi kel ski et al., 1999). Dur ing stress, changes in metab o lism help sup port the bio log i cal defences an indi vid ual uses to main tain homeo sta sis (Chrou sos and Gold, 1992; Wing field et al., 1998). These met a bolic alter a tions can result in neg a tive energy bal ance via a reduc tion in energy-sen si tive func tions such as growth or body mass main te nance (Chrou sos and Gold, 1992; Spencer et al., 2003; Spencer and Ver hulst, 2007; Laug ero and Mo berg, 2000a,b). Increased stress hor mone lev els are also often asso ci ated with an ele va tion in ener get i cally expen sive behav iors * Cor re spond ing author. E-mail address: k.spencer@bio.gla.ac.uk (K. Spencer). in avian spe cies, such as for ag ing, a strat egy thought to be ben e ficial in max i miz ing sur vival under stress ful con di tions (Ast hei mer et al., 1992; Breu ner et al., 1998; Ki tay sky et al., 2001). Whilst there is evi dence that day time metab o lism may be increased, stud ies of noc tur nal energy expen di ture have pro duced dif fer ing results. In birds stud ies have sug gested that exper i men tal admin is tra tion of CORT results in reduced noc tur nal metab o lism (Ast hei mer et al., 1992; But te mer et al., 1991). This phe nom e non is not man i fest as a reduc tion in basal or min i mum metab o lism, but a reduc tion in the var i a tion in met a bolic rate expe ri enced over night, reduc ing spon ta ne ous arousal bouts (Ast hei mer et al., 1992; But te mer et al., 1991). It has been sug gested that this is an adap tive strat egy to reduce daily energy needs whilst under stress. In con trast, sev eral mam ma lian stud ies have sug gested that chronic stress can cause increased arousal over night (Gro nli et al., 2004; Koe hl et al., 2002; Pa pale et al., 2005; Tiba et al., 2003). Stud ies to date exam in ing the role of stress in medi at ing meta bolic rate have been car ried out in adult ani mals and whilst recent evi dence sug gests that many spe cies can respond to external stress ors dur ing post-natal devel op ment (Blas et al., 2005; Bow er man et al., 2002; Ki tay sky et al., 1999, 2001; Love et al., 2003; Prav osudov and Ki tay sky, 2006; Sa i no et al., 2003; Sims and Holber ton, 2000; Sock man and Schwabl, 2001) there is a large gap in 0016-6480/$ - see front matter 2008 Else vier Inc. All rights reserved. doi:10.1016/j.ygcen.2008.09.007

K. Spencer, S. Ver hulst / General and Comparative Endocrinology 159 (2008) 250 256 251 our knowl edge of the ener getic costs of such responses in grow ing indi vid u als. In this study we aim to test the hypoth e sis that the met a bolic response of grow ing indi vid u als to ele vated stress hormone lev els fol lows the same pat terns as those seen in adult birds. Kita sky et al. (Ki tay sky et al., 2001) have shown that exper i mentally increas ing cor ti co ste rone by a fac tor of 1.8 above basal levels in juve nile black-headed gulls resulted in sig nifi cant increases in beg ging rate of between 60% and 180%. This was inter preted as an adap tive response to food stress by the chicks to gain access to more resources from the pro vi sion ing par ents, and indeed chicks with exper i men tally ampli fied beg ging rates received food at higher rates, a rela tion ship found in many spe cies (I aco vides and Evans, 1998; Kil ner, 1995; Ro y le et al., 2002; Sa i no et al., 2000). Thus grow ing birds exposed to cor ti co ste rone exhibit an ele vated diur nal metab o lism as seen in adult coun ter parts. There fore if young birds fol low the same ener getic pat terns over the whole day as adults and a damp ened over night metab o lism com pen sates for increased diur nal activ ity we would pre dict a reduced met a bolic rate over night to com pen sate for this increased expen di ture in animals that expe ri ence ele vated lev els of stress hor mones. How ever, all indi vid u als may not respond in the same way to ele vated stress hor mones. Sev eral stud ies have sug gested sig nifi cant sex dif fer ences in responses to devel op men tal con di tions (Arnold et al., 2007; de Ko gel, 1997; Ku di elka and Kirs ch baum, 2005; Liu et al., 2006; Mar tins, 2004; Mu el ler and Bale, 2007; Ra berg et al., 2005; Ver hulst et al., 2006). Females are more suscep ti ble to the neg a tive effects of devel op men tal stress in sev eral avian spe cies, includ ing the zebra finch, the spe cies used in this study (de Ko gel, 1997; Mar tins, 2004; Ver hulst et al., 2006), and there is some evi dence for increased met a bolic rates fol low ing stress ful rear ing envi ron ments (Ver hulst et al., 2006). How ever, the mech a nism under ly ing these sex-spe cific effects are still largely unknown. In addi tion the amount of sib ling com pe ti tion within a nest may restrict resources and have an addi tive effect under more stress ful con di tions. Brood size enlarge ments in avian spe cies have been shown to affect growth rates, immu no com pe tence, body con di tion and long term sur vival (de Ko gel, 1997; De er en berg et al., 1996; Eraud et al., 2008; Na guib et al., 2004; Soma et al., 2006). Since both sex and brood size have both been shown to be important in shap ing indi vid ual responses to devel op men tal stress we would hypoth e sise that females and birds from large broods would be more likely to exhibit sig nifi cant ele va tions in met a bolic rate under stress ful con di tions. Recently the role of early life con di tions in shap ing adult success and fit ness has been high lighted across a wide range of species, includ ing humans (Bate son et al., 2004; Buchanan et al., 2003; Gil, 2003; Kett er son et al., 1996; Met calfe and Mo na ghan, 2001; Mous saeu and Fox, 1998; Now ick i et al., 1998; Spencer et al., 2003, 2004). In par tic u lar the exper i men tal ele va tion of cor ti co ste rone dur ing early post-natal devel op ment has been shown to influ ence sev eral phe no typic traits later in life, such as sex ual sig nals, compet i tive abil ity, cog ni tion and fear-related behav iors (de Kloet et al., 2002; Ki tay sky et al., 2003; Spencer et al., 2003; Spencer and Ver hulst, 2007). One recent study has also linked early con di tions, in the form of sib ling com pe ti tion and brood size, to met a bolic rate in adult hood (Ver hulst et al., 2006), how ever, little is known about the short term effects of sib ling com pe ti tion on metab o lism. Thus it is pos si ble that post-natal expo sure to cor ti co ste rone could have long term effects on met a bolic rate. In this study we inves ti gated the imme di ate and long term effects of exper i men tal expo sure to the stress hor mone cor ti co sterone (CORT) dur ing the lat ter half of the nest ling period on standard met a bolic rate (SMR) and the var i a tion in SMR in the altricial bird spe cies the zebra finch (Ta e ni o py gia gut ta ta). The study uti lized genet i cally related sib ling pairs of birds, both male and female, from two manip u lated brood sizes (three or five chicks), where one received oral admin is tra tion of small amounts of CORT between the ages of 7 and 18 days post-hatch ing and the other received only the car rier solu tion (peanut oil) dur ing this period. This allowed us to deter mine the effects of sex, brood size and CORT lev els on metab o lism and deter mine any inter ac tive effects. 2. Meth ods 2.1. Cor ti co ste rone manip u la tion Birds in this study were also used in a pre vi ous study to deter mine the long term behav ioral effects of stress hor mones (for full details see Spencer and Ver hulst, 2007). In brief, adult zebra finches were ran domly paired (n = 23 pairs) and housed at the Bio log i cal Cen tre at the Uni ver sity of Gron in gen in 80 40 40 cm cages equipped with nest boxes and nest ing mate rial (hay). The birds were main tained at a tem per a ture of 20 24 C and a pho to pe riod of 14L:10D. Breeding pairs were pro vided with a com mer cial trop i cal seed mix ture (Teur lings, Dordr echt, The Neth er lands), water, shell grit, and cuttle fish bone ad libi tum. Pairs were also given egg food three times per week, until their off spring hatched when they received sup plemen tary egg food daily. Upon hatch ing first and sec ond hatched nestlings in each nest were indi vid u ally marked by col ored nail pol ish to each bird s toes. Brood sizes were stan dard ized to either three or five nes tlings (pop u la tion range 2 6 nes tlings) by add ing or removing nes tlings at 2 days of age (addi tional nes tlings in a nest were aged within 1 day of fos ter sib lings), ensur ing that two sib lings (first and sec ond hatched) remained in each nest that were genet i cally related to the par ents. This was done to deter mine any effects of brood size on SMR param e ters. When nes tlings reached 7 days of age exper i- men tal treat ments began. First and sec ond hatched chicks from each breed ing pair were assigned to one of two treat ments: cor ti co sterone admin is tra tion or con trol, count er bal anc ing across broods for chick hatch order to con trol for any potential dif fer ences in basal cor ti co ste rone lev els (Schwabl, 1999). As described in Spencer and Ver hulst (2007) birds in the cor ti co ste rone group were dosed with 0.0125 mg of cor ti co ste rone daily, dis solved in peanut oil (con centra tion 0.25 mg/ml) via two 25 ll doses (at least 6 hours apart) from the age of 7 18 days of age. This dos ing schedule results in a short term increase in plasma cor ti co ste rone lev els (Spencer and Ver hulst, 2007). To deter mine this sin gle blood sam ples were taken a subsam ple of birds (n = 32) at dif fer ent times fol low ing dos ing on day 12 post-hatch ing (details in Spencer and Ver hulst, 2007). Fol lowing dos ing CORT lev els peaked after 10 min and the lev els of CORT expe ri enced by our nes tlings fell within the nat u ral range of nest ling zebra finch responses to a stan dard ized cap ture han dling restraint pro to col at 16 days post-hatch ing (Wada et al., 2008) and we have recently col lected data to sug gest that the peak response to a standard ized stressor does not dif fer between nes tlings of 12 and 16 days of age (Spencer et al., unpub lished data). The sam pling regime used to deter mine the effects of our treat ment meant that a sub-sam ple of birds (n = 16) had been bled the day before our met a bolic measure ments, how ever, all sib lings were mea sured on the same day and these birds are spread equally across sex and brood size. Control birds were dosed with the same amount of the peanut oil car rier solu tion at the same time as their sib ling. All manip u la tions ceased at the age of 18 days. All birds had their mass recorded at reg u lar intervals, par tic u larly dur ing each met a bolic rate mea sure ment at day 12 and 55 65 days. The sex of each bird was deter mined via the presence of sex u ally dimor phic adult plum age, which begins to appear after 35 days of age. Due to mor tal ity two birds were removed from the anal y sis, leav ing us with a final sam ple size of 44 birds (from 23 sib ling pairs); a fur ther break down of sam ple sizes can be seen on the rel e vant fig ures. All pro ce dures were car ried out fol low ing peer review and approval from the eth i cal com mit tee for ani mal exper i- ments of the Uni ver sity of Gron in gen (under license D4299).

252 K. Spencer, S. Ver hulst / General and Comparative Endocrinology 159 (2008) 250 256 2.2. Met a bolic rate mea sure ments Met a bolic mea sure ments were car ried out as described in Ver hulst et al. (2005). Briefly, each bird was removed from its nest between 18.30 and 19.30 h, 1.5 2 h prior to the end of the light period and placed inside a dark 2 l Plex i glas box equipped with a perch at a tem per a ture rep re sen ta tive of the pre vail ing ambi ent tem per a ture. At 11 13 days of age this meant main tain ing a tem per a ture of 30 C, the mean tem per a ture within the nest cup. The sec ond trial was con ducted when birds were between 55 and 65 days of age, hence the tem per a ture was set at 22 C to mimic their hold ing cage con ditions. Although the age of birds var ied in the met a bolic tri als across sib ling pairs, each pair of birds was mea sured on the same night to con trol for potential age effects. The air flow rate was con trolled by mass-flow con trol lers (5850S, Brooks, Ri jswijk, The Neth er lands), that were cal i brated with a bub ble flow meter, set to deliver 16 l h 1. In- and out-flow ing air was dried by pass ing through a molec u lar sieve (3 Å, Merck, Darms tadt, Ger many). Gas anal y sis was done using a para mag netic O2-ana lyser (Ser vo mex Xen tra 4100, Crow bor ough, UK) and CO2-ana lyser (Ser vo mex 1440). The sys tem was cal i brated before each mea sure ment ses sion using two three-digit precision gas mix tures of 20.0% O 2 /0% CO 2 and 21.0% O 2 /1.0% CO 2 in N 2. CO 2 and O 2 mea sure ments were recorded at 7 min inter vals (between 81 and 87 record ings were taken over the mea sure ment period) for approx i mately 10 h, which is the nor mal length of the dark period expe ri enced by these birds in this exper i ment. The rate of oxy gen con sump tion was cal cu lated from these mea sure ments and converted to the energy equiv a lent, while cor rect ing for the respi ratory quo tient, accord ing to Brody (Brody, 1945). SMR was taken to be the min i mum value of a 30 min run ning mean for each indi vidual. In addi tion the stan dard devi a tion of the min i mum 30 min running mean was also cal cu lated to deter mine var i abil ity in noc tur nal energy expen di ture (here called stan dard devi a tion of SMR), in line with But te mer et al. (1991). Body mass was mea sured before and after res pi rom e try. 2.3. Sta tis ti cal anal y sis All data were ana lyzed using Lin ear Mixed Mod els (SPSS, ver sion 13.0), with cage of birth entered as random effect. SMR and the stan dard devi a tion of SMR were used as depen dent vari ables in each anal y sis. For all anal y ses data from the two tri als were com bined to per form repeated-mea sures mod els with indi vid ual nested within cage of birth entered as an addi tional random fac tor. Fixed vari ables in each model were: brood size (three or five), sex, treat ment (con trol or CORT), age (11 13 or 55 65 days) and hatching order (first or sec ond). In addi tion mass (mean of val ues taken before and after each res pi rom e try mea sure ment) was entered as a covar i ate. Step wise dele tion of non-sig nifi cant non-hier ar chi cal terms was employed. All resid u als were checked for nor mal ity. 3. Results 3.1. Noc tur nal metab o lism The anal y sis of SMR showed a sig nifi cant inter ac tion between age, sex and brood size (F 1,55.3 = 6.20, p = 0.015), where females from both brood sizes and males from small brood sizes (con tain ing three chicks) showed sim i lar small decreases in SMR with age, but in males from large brood sizes (con tain ing five chicks) SMR was main tained over time (Fig. 1). There was also a sig nifi cant main effect of age (F 1,34.1 = 4.62, p = 0.034), indi cat ing in this case that the inter cepts of the two met a bolic tri als dif fer sig nifi cantly. Birds from sec ond hatched eggs also exhib ited sig nifi cantly ele vated met a bolic rates, inde pen dent of mass as the model con trolled for this, show ing an increase of 6% over birds from first hatched eggs There was a sig nifi cant inter ac tion between age and treat ment (F 1,36.9 = 5.27, p = 0.03; Fig. 2), as CORT admin is tra tion had immedi ate effects on the var i abil ity of noc tur nal metab o lism but no long term effects, with CORT-fed birds show ing a trend towards ele vated stan dard devi a tions of SMR dur ing the nest ling period (t (21) = 1.4, P = 0.08). Brood size also inter acted with treat ment and age (F 1,37.9 = 4.82, p = 0.03, Fig. 3), sug gest ing that the inter action between age and treat ment was driven by the effects seen in larger brood sizes, whilst those birds from small brood sizes show sim i lar responses to the treat ment. Again there was a sig nifi cant main effect of age (F 1,36.9 = 20.7, p < 0.0001), and a non-sig nifi cant trend towards an effect of brood size (F 1,22.9 = 3.44, p = 0.07). There was also a sig nifi cant inter ac tion between sex and treat ment (F 1,38.7 = 7.15, p = 0.011; Fig. 4), with CORT-fed females show ing signifi cantly more var i abil ity in SMR (t 39 = 2.0, p = 0.05), whilst males showed no met a bolic responses to CORT admin is tra tion (t 39 = 1.1, p = 0.27). There was also a non-sig nifi cant trend towards a threea b Fig. 1. The effect of manip u lated brood size on the stan dard met a bolic rates (W) of male and female zebra finches mea sured at (a) 11 13 and (b) 55 65 days posthatch ing: age brood size sex inter ac tion: F 1,55.3 = 6.20, p = 0.015. Sam ple sizes are shown within each bar. (F 1,36.9 = 4.62, p = 0.036). Mean mass was sig nifi cantly pos i tively related to SMR (F 1,33.3 = 31.7, p < 0.0001). How ever, there were no effects of treat ment on SMR and no other sig nifi cant inter ac tions (p > 0.4). There were no dif fer ences in the masses of sib lings at either 11 13 or 55 65 days of age (F 1,40 = 0.34, p = 0.56, F 1,40 = 0.03, p = 0.81, respec tively), how ever, birds fed with CORT in this pop u lation did show reduced mass com pared to con trols at the end of the treat ment period (18 days of age, F 1,18.5 = 31.91, p < 0.001, sta tis tics from Spencer and Ver hulst, 2007). 3.2. Var i a tion in SMR

K. Spencer, S. Ver hulst / General and Comparative Endocrinology 159 (2008) 250 256 253 a Fig. 2. The effects of age and CORT treat ment on the stan dard devi a tion in SMR (W), sug gest ing that stress hor mones admin is tered dur ing post-natal devel op ment have only short term effects on metab o lism. Treat ment age inter ac tion: F 1,36.9 = 5.27, p = 0.027. Sam ple sizes are shown above treat ment. b term inter ac tion (age treat ment sex inter ac tion: F 1,38.1 = 3.86, p = 0.056), con firm ing that these treat ment effects are mainly due to changes in female metab o lism dur ing the trial at 12 days of age. There were no effects of hatch ing order on SMR var i abil ity (F 1,37.4 = 0.26, p = 0.63). 4. Dis cus sion 4.1. Short term effects on metab o lism Our results dem on strate that devel op men tal expo sure of avian nes tlings to glu co cor ti coid hor mones can have sig nifi cant short term effects on stan dard metab o lism, result ing in increased var i- abil ity over night. Whilst pre vi ous work has pro vided evi dence for met a bolic changes in response to stress in adults, to our knowl edge this is the first study to exam ine this directly in grow ing indi vid u- als. This increase in met a bolic var i abil ity is con trary to our hypothe sis that we would find the same rela tion ship found in adult birds, i.e. that ele vated CORT lev els decrease noc tur nal metab o lism by reduc ing the amount of arousal events over night (Ast hei mer et al., 1992; But te mer et al., 1991). It has been sug gested that this reg u lates daily energy bud gets and re-coups reserves for the follow ing day in order to avoid or over come the stressor (Ast hei mer et al., 1992; But te mer et al., 1991). Our data sug gest that growing birds may be more sen si tive to stress than adults, or may be less able to reg u late their energy expen di ture over a 24 h period. Our results are, how ever, more con sis tent with work on mam mals, that sug gests chronic stress causes increased noc tur nal arous als, i.e. increased var i abil ity in met a bolic rate, which have been linked to increased sleep frag men ta tion (Gro nli et al., 2004; Koe hl et al., 2002; Pa pale et al., 2005; Tiba et al., 2003). Such sleep dis tur bances are known to increase over night energy expen di ture, al lo stat ic load and energy imbal ance (Bon net et al., 1991; McE wen, 2006). We did not mea sure sleep frag men ta tion in this study and therefore can draw no firm con clu sions about the effects of our treatments on sleep param e ters and fur ther work would be required to deter mine this. Although birds from the CORT-fed group in our study did not show reduced masses at the time of the met a bolic mea sure ments, body mass in these birds was sig nifi cantly reduced at the end of the treat ment period, at 18 days of age (Spencer and Fig. 3. The effect of CORT treat ment and brood size (n = 3 or 5 nes tlings) on the var i a tion in SMR (W) dur ing two tri als at age (a) 11 13 and (b) 55 65 days: treatment age brood size inter ac tion: F 1,37.9 = 4.82, p = 0.03. Sam ple sizes are shown within each bar. Fig. 4. The effect of CORT admin is tra tion on the var i abil ity of SMR in male and female zebra finches. Treat ment sex inter ac tion: F 1,38.7 = 7.15, p = 0.011. Females showed a sig nifi cant increase in var i abil ity fol low ing CORT treat ment (t 39 = 2.0, p = 0.05), whereas males showed no response (t 39 = 1.1, p = 0.27). Sam ple sizes are shown within each bar.

254 K. Spencer, S. Ver hulst / General and Comparative Endocrinology 159 (2008) 250 256 Ver hulst, 2007). This sug gests that repeated stress for a pro longed period may have an incre men tal effect on growth. Our exper i men tal manip u la tion mim ics a nat u ral increase in glu co cor ti coids fol low ing a stressor. This repeated increase in stress hor mones would have the increased al lo stat ic load of the indi vid ual, i.e. the cumu la tive costs of main tain ing the inter nal stabil ity of the ani mal (Kor te et al., 2005; McE wen, 2006; Wing field, 2005). The observed responses to the ele va tion in stress hor mones could then be part of an al lo stat ic response to stress ful con ditions to reduce over all al lo stat ic load. How ever, it is di f icult to see how an increase in met a bolic var i abil ity and poten tially over night arousal would con fer any met a bolic advan tage on the ani mal and restore the ani mal to its nor mal state. It is more likely that the stressor imposed by our manip u la tions resulted in al lo stat ic overload (type 2), which did not trig ger trans fer to an emer gency life his tory stage, but instead con ferred sig nifi cant con straints on the indi vid ual, depress ing growth rates (Kor te et al., 2005; Wing field, 2005). How ever, fur ther work is required to iden tify the pos si ble adap tive sig nifi cance of the met a bolic changes seen in this study. Expo sure to CORT did not affect all indi vid u als in this study. We found that CORT-fed indi vid u als from exper i men tally enlarged brood sizes had sig nifi cantly more var i abil ity in SMR. Brood size enlarge ments in this spe cies have been shown to affect growth rates (de Ko gel, 1997; De er en berg et al., 1996), and pro duce long term effects on sur vival post-inde pen dence (de Ko gel, 1997; De er en berg et al., 1996). Sib ling rivalry would have been more intense in these broods, along with a pos si ble reduc tion in food intake. Thus the results sug gest that there are addi tive effects of stress at work here, and are in line with our pre dic tion that brood size would show an inter ac tion with ele vated stress hor mones. It is pos si ble that birds in larger broods had ele vated CORT lev els, which might have aug mented our exper i men tal pro ce dures, but we have no data to eval u ate this hypoth e sis. Our results sug gest that birds in large broods would suf fer larger met a bolic changes dur ing periods of fur ther food short ages or another exter nal stressor than birds in small broods. We also detected sex dif fer ences in the met a bolic response to glu co cor ti coid expo sure, as pre dicted. Although there is no sex ual size dimor phism in zebra finches, pre vi ous stud ies have sug gested that females may be more sus cep ti ble to nutri tional stress through either food restric tion or brood size manip u la tion dur ing develop ment, result ing in decreased sur vival or altered metab o lism (de Ko gel, 1997; Mar tins, 2004; Ver hulst et al., 2006). Inter est ingly Mar tins (2004) showed that females grew more slowly than males when under iden ti cal exper i men tal food restric tion in the absence of sib ling com pe ti tion. Since our results sug gest that females do not have a damped noc tur nal energy strat egy to com pen sate for any increases in day time activ ity, such as beg ging for food, this may pro vide a mech a nism by which grow ing females under chronic stress show reduced growth rates. The fact that males in this study did not seem to respond to the exper i men tal treat ment, but were more sen si tive to the amount of sib ling com pe ti tion dur ing the nest ling stage (brood size), may sug gest sex-spe cific mech a nisms for cop ing with stress ful envi ron ments. Pre vi ous stud ies in birds that have uti lized exper i men tal manipu la tions of glu co cor ti coids have mainly used meth od ol o gies that cause ele va tions in base line lev els of CORT, such as silas tic implants (Ast hei mer et al., 1992; But te mer et al., 1991). These tech niques would mean that base line lev els were prob a bly ele vated over the whole 24 h period, includ ing the noc tur nal period when met a bolic mea sure ments were taken. In con trast, our feed ing regime only caused short term increases in CORT for less than 2 h after dosing (Spencer and Ver hulst, 2007). This dif fer ence in meth od ol o gies may explain the dif fer ences seen in over night metab o lism. However, we assert our exper i men tal pro ce dure is bio log i cally more rel e vant when aim ing to mimic repeated acute stress ors, such as pred a tor attacks or dis tur bance, in par tic u lar because strong fluctu a tions in plasma lev els are inher ent to glu co cor ti coid hor mones. How ever, we still know very little about nest ling responses to such nat u ral stress ors and more work is required in this area. One study in a lab o ra tory pop u la tion of rats has sug gested that acute stressors applied at either the onset or con clu sion of the light period always show their effects on sleep frag men ta tion within the dark cycle (Koe hl et al., 2002). This con firms that CORT can have delayed effects on metab o lism and sleep and sup ports our inter pre ta tion of the cur rent results. 4.2. Long term effects on metab o lism We found no long term effects of exper i men tal CORT admin istra tion on the var i abil ity of SMR. How ever, SMR tended to decrease over the two res pi rom e try tri als in all birds from small brood sizes and in females from larger broods, despite a decrease in ambient tem per a ture, which would nor mally be asso ci ated with an increase in met a bolic rate (Gav ri lov, 1996). Whilst sev eral stud ies have shown that rest ing metab o lism is highly repeat able in avian spe cies (Bech et al., 1999; Ron ning et al., 2005; Vez in a and Williams, 2005), they have not yet com pared metab o lism dur ing the nest ling stage with later stages of devel op ment. In this study SMR com par i sons between the two met a bolic tri als revealed non-signifi cant repeat abil ity in both of our met a bolic param e ters (SMR: F 43,44 = 0.84, p = 0.72; sd: F 43,44 = 1.17, p = 0.30). An age-related decline in stan dard or rest ing metab o lism has, how ever, been well doc u- mented in mam ma lian spe cies, includ ing humans (Hughes et al., 1998; Neu ha us er-bert hold et al., 2000; Speak man et al., 2003; Van Pelt et al., 1998, 2001) and is thought to relate to changes in body com po si tion (Hughes et al., 1998). How ever, these stud ies tend to mea sure age-related changes dur ing adult hood and this neg a tive rela tion ship is not universal. In one recent avian study has provided evi dence for a positive rela tion ship between age and rest ing met a bolic rate (Pis and Lus nia, 2005). In addi tion we found a sig nifi cant effect of hatch ing order on SMR, regard less of age and inde pen dent of mass, with sec ond hatched chicks exhib it ing ele vated met a bolic rates com pared to their first hatched sib lings. Pre vi ous work in kes trels has sug gested that hatch ing order can affect metab o lism, with first hatched chicks show ing ele vated daily energy expen di tures (Masse min et al., 2003) and last hatched chicks show ing sig nifi cantly lower resting metab o lism com pared to their older sib lings (Masse min et al., 2002), which are at odds with our results. How ever, first and second hatched chicks have been shown to have dif fer en tial lev els of both base line and stress-induced CORT dur ing the nest ling period, invari ably with first hatched chicks show ing ele vated hor mone lev els (Schwabl, 1999; Love et al., 2003). There fore endog e nous lev els of CORT may dif fer across hatch ing order in our pop u la tion and play a role in medi at ing SMR both dur ing the nest ling period and in later life. How ever, it is likely that these birds dif fer in several other aspects and fur ther work would be required to elu ci date the mech a nisms under ly ing the effects seen in this study. There were also per sis tent effects of brood size on SMR, as males from nests with a brood size of five failed to show the age-related decline in met a bolic rate seen in other birds in this and other studies (Hack, 1997; Klau sen et al., 1997), result ing in ele vated rates in some males at sex ual matu rity. A recent study using the zebra finch has also shown long term effects of brood size on stan dard met a bolic rate (Ver hulst et al., 2006); how ever, in con trast to our find ings they reported stron ger effects on metab o lism in female birds. Very sim i lar meth od ol o gies were used to quan tify SMR in these two stud ies, but in the ear lier study birds were mea sured when they were over one year old. How ever, both stud ies sug gest that sib ling com pe ti tion can have long term effects on metab o lism in later life.

K. Spencer, S. Ver hulst / General and Comparative Endocrinology 159 (2008) 250 256 255 In sum mary, our results show that expo sure to glu co cor ti coid hor mones can have sig nifi cant effects on the var i abil ity of noc tur nal metab o lism, spe cifi cally in broods that had expe ri enced height ened sib ling com pe ti tion. This could have impor tant con sequences for energy bal ance and growth over the nest ling period, how ever, the true fit ness costs of such changes to met a bolic rate require fur ther inves ti ga tion. Whilst we found no long term effects of our exper i men tal treat ments, our results also sug gest that increased sib ling com pe ti tion can ele vate stan dard metab o lism in the long term. Acknowl edg ments We thank Henk Vis ser who pro vided crit i cal assis tance with license appli ca tions. We also thank Mar ti jn Sa lo mons for assis tance with exper i men tal pro to cols and Arthur Gold smith for guid ance on assay pro to cols and lab o ra tory use. 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