Hematological changes associated with egg production: direct evidence for changes in erythropoiesis but a lack of resource dependence?

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1 2960 The Journl of Experimentl Biology 211, Pulished y The Compny of Biologists 2008 doi: /je Hemtologicl chnges ssocited with egg production: direct evidence for chnges in erythropoiesis ut lck of resource dependence? Emily C. Wgner*, Christine A. Stles nd Tony D. Willims Deprtment of Biologicl Sciences, Simon Frser University, 8888 University Drive, Burny, British Columi, Cnd V5A 1S6 *Author for correspondence t present ddress: Womenʼs Helth Reserch Institute, Provincil Helth Services Authority, Room E Ok Street (Box 42), Vncouver, British Columi, Cnd V6H 3N1 (e-mil: ewgner3@cw.c.c) Accepted 14 July 2008 SUMMARY Reductions in hemtologicl prmeters mong lying irds re well reported, ut the cuse of this nemi is not known. We tested specific predictions generted from severl, non-mutully exclusive hypotheses for mechnisms underlying reproductive nemi ssocited with egg production (hemodilution, trnsient suppression of erythropoiesis, resource dependence) in reltion to (1) the time-course of development nd recovery from nemi, (2) chnges in specific hemtologicl trits, nd (3) the effect of diet qulity, in femle zer finches (Teniopygi guttt). Femle zer finches showed mrked decreses in hemtocrit (~6%), red lood cell counts (~8%), nd plsm hemogloin concentrtion (~9%) during egg production, even on high-qulity d liitum diet, consistent with n effect of hemodilution ssocited with yolk precursor production. However, our results provide strong support for the hypothesis tht erythropoiesis is trnsiently suppressed during egg-lying nd tht the recovery from nemi is reltively long-lsting, extending through incution nd htching periods. Decresed hemtocrit, red lood cell counts, nd hemogloin concentrtion did not recover t clutch completion, ut showed evidence of recovery to seline pre-reeding levels t htching. More importntly, there ws significnt time-dependent vrition in the proportion of reticulocytes, which incresed t clutch completion ut peked t htching dys fter clutch completion, nd in men red lood cell volume, which showed significnt increse t clutch completion; consistent with enhnced production nd relese of lrger immture cells into the circultion following suppression of erythropoiesis. Finlly, we found no evidence for resource dependence of nemi ssocited with egg production in reltion to diet qulity, i.e. exogenous lipid nd protein resources ville to the lying femle. This study demonstrtes tht trnsient suppression of erythropoiesis nd, susequently, incresed reticulocytosis, re key components of reproductive nemi in egg-lying femles. Key words: nemi, cost of reproduction, egg production, erythropoiesis, estrogen, zer finch. INTRODUCTION Although physiologicl mechnisms underlying costs of reproduction remin poorly understood (Zer nd Hrshmn, 2001; Hrshmn nd Zer, 2007), this trde-off etween current reproduction nd future fecundity nd/or survivl hs trditionlly een ssumed to involve relloction of resources mong different, competing physiologicl systems (Sterns, 1992). More recently it hs een suggested tht the reproductive process itself or the regultory (physiologicl) networks controlling reproduction (e.g. Prtridge et l., 2005; Hrshmn nd Zer, 2007) might induce costs of reproduction. Hormones re prticulrly strong cndidtes for generting s well s regulting such trde-offs ecuse hormones cn hve pleiotropic effects, oth positive nd negtive, on multiple physiologicl systems (e.g. Finch nd Rose, 1995; Ketterson nd Noln, 1999; Willims et l., 2005; Zer et l., 2007). Here we focus on one potentil physiologicl mechnism tht might medite costs of reproduction: the development of nemi during egg production in irds (Willims et l., 2004; Willims, 2005). Anemi reduction in hemtocrit, hemogloin nd red lood cell numer (Cmpell nd Ellis, 2007) during egg production hs een documented in numerous studies [(e.g. degrw et l., 1979; Jones, 1983; Morton, 1994; Gythri nd Hegde, 2006; Wgner et l., 2008) nd references therein], nd my persist into lter stges of incution nd chick rering in some cses (Willims et l., 2004). In ddition, Klmch et l. (Klmch et l., 2004) showed tht experimentlly incresing egg production in femle gret skus (Stercorrius sku) incresed nemi, in terms of greter reduction in hemtocrit nd red lood cell numer compred with control femles. Since nemi should reduce totl oxygen crrying cpcity of the lood nd compromise eroic performnce, this might explin negtive effects of incresed egg production effort on susequent reproductive stges (i.e. incution nd chick rering) tht hve een widely reported in irds (e.g. Monghn et l., 1998; Nger et l., 2001). Severl potentil mechnisms hve een proposed to explin the development of nemi during egg production. Decresed hemtocrit might e n indirect effect of estrogen-dependent heptic production of yolk precursors nd moiliztion of clcium ions (Morton, 1994; Slvnte nd Willims, 2002), osmoticlly ctive compounds tht re trnsported in the lood t high concentrtions during egg production. This in turn my trigger n increse in plsm volume due to osmotic movement of wter from extr-cellulr spces into the lood (e.g. hemodilution) to mintin plsm osmolrity or viscosity t constnt level (Reynolds nd Wldron, 1999), which would decrese hemtocrit (red lood cells per unit plsm volume) ut not totl cell numer. Some uthors hve proposed tht the reduction in hemtocrit reflects trnsient suppression of erythropoiesis (e.g. red lood cell production) during egg production in order to redirect energy to meet the incresed metolic demnds of the reproductive orgns (Ronld et l., 1968), or ecuse essentil

2 Hemtologicl chnges during egg lying 2961 fctors required for erythropoiesis re preferentilly llocted to the production of egg components (Jones, 1983; Gythri nd Hegde, 2006; Ksprzk et l., 2006). Alterntively, development of nemi might represent direct, negtive pleiotropic effect of estrogen, which is present t high levels during egg production nd hs essentil reproductive functions (Klmch et l., 2004; Willims et l., 2004; Willims et l., 2005). Estrogens inhiit the differentition, prolifertion nd survivl of white nd red lood cell precursors in the one mrrow (Bloel nd Orkin, 1996; Medin et l., 2000; Perry et l., 2000). Blocking estrogen receptors using the nti-estrogen tmoxifen inhiits the development of nemi in egg-lying irds supporting role for estrogen-dependent suppression of erythropoiesis in nemi (Wgner et l., 2008). The cndidte mechnisms tht hve een proposed involve very different predictions out (1) the time-course of development nd recovery from nemi, (2) whether specific chnges would occur mong suset or ll hemtologicl vriles (hemtocrit, hemogloin concentrtion, red lood cell size nd numer, proportion of immture red lood cells or reticulocytes; see Tle 1), nd (3) the extent to which development of nemi should e influenced y resource vilility or diet qulity. In the present study, we tested predictions generted from these different, non-mutully exclusive hypotheses for the cusl mechnisms underlying nemi ssocited with egg production using femle zer finches (Teniopygi guttt; see Tle 1). Specificlly, if hemodilution is solely responsile for the oserved decrese in hemtocrit, we predicted tht, (1) red lood cell numer nd hemogloin per unit plsm volume would lso decrese during egg production, ut tht these chnges would e reversed t clutch completion when plsm yolk precursor concentrtion returned to non-reeding levels (Slvnte nd Willims, 2002), nd (2) there would e no chnges in men red lood cell size in reltion to cell ge [e.g. newly produced red lood cells re lrger thn mture cells (Cmpell nd Ellis, 2007)] or in the proportion of reticulocytes, since dilution per se would not chnge rtes of red lood cell turnover (production nd degrdtion of cells). By contrst, if nemi involved trnsient, estrogen-dependent suppression of erythropoiesis, we predicted tht (1) decresed hemtocrit, cell numer nd hemogloin concentrtion would not recover t clutch completion since it tkes 7 14 dys for regenertive erythropoiesis to restore red lood cell numers following experimentlly induced nemi (Domm nd Ter, 1946; Clrk et l., 1988), nd (2) there would e mrked increse in immture red lood cells (reticulocytes) post-lying nd corresponding increse in men cell volume [i.e. reticulocytes re lrger thn mture cells (Cmpell nd Ellis, 2007)]. Finlly, if nemi during egg production is resource dependent, we predicted tht femles on low-qulity diet (e.g. protein nd lipid deficient) would show lrger decreses in hemtocrit, red lood cell counts nd hemogloin concentrtion compred with femles on high-qulity diet. MATERIALS AND METHODS Study species nd husndry Zer finches (Teniopygi guttt Vieillot) were housed under controlled environmentl conditions (temperture C, humidity 35 55%, constnt light schedule of 14 h:10 h L:D, lights on t 07:00h). All irds received mixed seed diet (pnicum nd white millet 1:1; pproximtely 11.7% protein, 0.6% lipid nd 84.3% crohydrte), wter, grit nd cuttlefish one (clcium) d liitum, nd multivitmin supplement in the drinking wter once per week. All experiments nd niml husndry were crried out under Simon Frser University Animl Cre Committee permit (657B- 96) following guidelines of the Cndin Committee on Animl Cre. Prior to the experiment, ll irds were housed in sme-sex cges, ut were not visully or cousticlly isolted from the opposite sex. The femles selected for this experiment were pproximtely 8months of ge nd hd een red twice previously t 4 nd 6 months of ge, i.e. they were ll experienced reeders. Femles were rndomly pired with n experienced mle, nd reeding pirs were housed individully in cges ( cm) equipped with n externl nest ox ( cm). Body mss (±0.001g) nd trsus length (±0.01mm) of oth irds ws recorded t the time of piring. Nest oxes were checked dily etween 09:00 nd 11:00 to otin dt on lying intervl (the numer of dys elpsed etween piring nd lying of the first egg), egg sequence, egg mss (±0.001g), nd clutch size. Clutches were considered complete when no dditionl eggs were produced over two consecutive dys. Experimentl protocol Twenty-nine reeding pirs were rndomly ssigned to either lowqulity (N=14) or high-qulity diet tretment (N=15). Between piring nd clutch completion, femles on the low-qulity diet continued to receive the stndrd seed diet d liitum (see ove), while those on the high-qulity diet received the stndrd seed diet plus dily egg food supplement (6gdy 1, 20.3% protein:6.6% lipid). Initil oservtions showed tht htching success ws extremely low on the low-qulity diet, confirming tht this diet ws poor qulity, so ll pirs receiving this diet were seprted nd returned to sme-sex holding cges t clutch completion. As such, we present dt from the htching nd chick-rering periods for femles receiving the high-qulity diet only: reeding pirs receiving the high-qulity diet were permitted to incute eggs nd rer chicks to fledging, with egg-food provided gin during the chick-rering period. Pirs were left undistured from clutch completion until the htching period, t which point nest oxes were checked dily to determine htching success per clutch. Immeditely fter htching, Tle 1. Summry of predictions generted from ʻhemodilutionʼ versus ʻsuppression of erythropoiesisʼ hypotheses for chnges in hemtologicl prmeters during the femle reeding cycle Trit Hemodilution Suppression of erythropoiesis Hemtocrit Decrese t stge Decrese t stge Recovery t clutch completion Recovery fter clutch completion Red lood cell numer Decrese t stge Decrese t stge Recovery t clutch completion Recovery fter clutch completion Hemogloin concentrtion Decrese t stge Decrese t stge Recovery t clutch completion Recovery fter clutch completion Red lood cell volume No chnge Increse t clutch completion or htching (incresed proportion of lrger, immture reticulocytes) Proportion of reticulocytes No chnge Increse t clutch completion or htching (incresed reticulocytosis)

3 2962 E. C. Wgner, C. A. Stles nd T. D. Willims chicks were weighed nd mrked with non-toxic dye to indicte htch order, nd then individully nded t 8dys of ge. The mss of ech chick ws recorded t 7, 10, 14 nd 21dys post-htch to monitor growth rtes. At 30dys of ge, finl rood size for ech nest ws recorded, nd weight, trsus length, nd ill length of ech chick ws mesured. After 3month rest period, the sme mtched pirs were red gin under the opposite diet regime thn previously ssigned, such tht repeted mesures dt ws otined for 23 pirs tht initited egg-lying on oth diet regimes (of the originl 29 femles two died in the intervening rest period (oth from the highqulity diet tril) nd four femles either did not ly eggs or lid only one egg nd were eliminted from the second low-qulity diet tril). Blood smpling nd hemtologicl nlysis To monitor hemtologicl prmeters cross the reeding cycle, femle irds were lood smpled t five intervls. (1) pre-reeding t piring (N=29); (2) egg-lying dy of lying of first egg (N=29); (3) clutch completion fter two consecutive dys without lying n egg (N=25); (4) htching the dy the first chick htched (N=18); nd (5) fledging on verge 21 dys post-htching (N=12). For the diet-qulity nlysis we used lood smples otined for 23 femles t prereeding, the stge, nd clutch completion only. All lood smples (~50μl) were collected within 3min of cpture from the rchil vein (to void potentil cpture-relted stress effects) etween the hours of nd Hemtologicl vriles were mesured with stndrd techniques developed for humn lood nd commonly used on irds (Cmpell nd Ellis, 2007). Hemtocrit (Hct; %) ws mesured with digitl clipers (±0.01 mm) following centrifugtion of whole lood for 3min t 13,000g. Hemogloin (H; gdl 1 whole lood) ws mesured using the cynomethemogloin method (Drkin nd Austin, 1932) modified for use with microplte spectrophotometer (BioTek Powerwve 340, BioTek Instruments, Winooski, VT, USA), using 5μl whole lood diluted in 1.25ml Drkin s regent (D5941 Sigm-Aldrich Cnd, Okville, Ontrio, Cnd) with sornce mesured t 540 nm. Intr- nd inter-ssy coefficients were 1.7% nd 3.9%, respectively. Erythrocyte counts (RBC; numer of cells 10 6 μl 1 ) were determined from duplicte smples (1 μl lood diluted 1/200 with modified Ntt nd Herrick s solution (Ntt nd Herrick, 1952; Roertson nd Mxwell, 1990) with n improved Neuuer hemocytometer (Fisher Scientific, Ottw, Ontrio, Cnd). The verge vrition mong duplicte RBC smples from the sme ird ws 6.9%, nd mesurement error (determined from repeted smpling) ws 8.9%, which is to e expected with this technique (Cmpell nd Ellis, 2007). From these mesurements we clculted men red cell volume (MCV; femtolitres or fl) using the formul Hct/RBC 10=MCV (Cmpell nd Ellis, 2007). The proportion of reticulocytes (% Ret=numer of immture red lood cells/totl red lood cells 100) ws estimted from whole lood smers fter suprvitl stining with new Methylene Blue (R4132, Sigm Aldrich Cnd, Okville, Ontrio, Cnd). A totl of 1000 red lood cells were counted per slide, nd reticulocytes were distinguished from mture erythrocytes y their reltively lrger size nd less condensed chromtin (Cmpell nd Ellis, 2007). Red lood cells were clssified s reticulocytes if t lest five reticulum (RNA) ggregtions were visile in the cytoplsm or if distinct ring of reticulum ws surrounding the nucleus (Fernndez nd Grindem, 2006). The sme individul counted ll lood smers (E.C.W.), nd slides were rndomly coded prior to nlysis so tht the exminer ws lind to the identity of the femle nd reproductive stge eing scored. If lood smples collected t vrious stges were of insufficient volume to permit ll nlyses listed ove, priority ws given to mesuring hemtocrit (finl smple sizes re listed in Tle 2). Sttisticl nlysis All sttisticl nlyses were crried out using SAS softwre version 9.1 (SAS 2003). Seprte repeted-mesures nlyses were performed to (1) exmine vrition in ody mss nd hemtologicl prmeters cross the entire reeding cycle in femles on the high qulity diet (pre-reeding,, clutch completion, htching nd fledging stges), nd (2) exmine the effect of diet qulity on chnges in ody mss nd hemtologicl prmeters during egg-production (pre-reeding, nd clutch completion). To exmine temporl vrition in ody mss nd hemtologicl prmeters, we used repeted-mesures mixed liner models (MIXED procedure) with reproductive stge included in the model s fixed effect nd individul s rndom effect. For the second nlysis (effect of diet qulity), diet type nd diet stge were lso included s fixed effects in the model. For ll repeted mesures nlyses, the denomintor degrees of freedom for tests of fixed effects nd post-hoc tests were computed using the Kenwrd-Roger method s recommended y Littell et l. (Littell et l., 2006). The etween- nd individulvrince components (e.g. rndom effects) were clculted nd evluted vi comprison with Gussin distriution expected if the vrince/covrince ws equl to zero. Post-hoc tests for differences etween mens were corrected for multiple comprisons using the Tukey Krmer djustment formul. Clutch size ws the only vrile tht ws not pproximtely norml in distriution Tle 2. Smple sizes for hemtologicl nlyses in ech experiment Pre-reeding stge Htching Fledging A. Experiment 1: temporl vrition in hemtologicl prmeters Body mss Hemtocrit Hemogloin Red lood cell numer Men cell volume Reticulocyte counts B. Experiment 2: effect of diet qulity on temporl vrition in hemtologicl prmeters Body mss Hemtocrit Hemogloin Red lood cell numer Men cell volume Reticulocyte counts

4 Hemtologicl chnges during egg lying 2963 (Kruskl Wllis test; UNIVARIATE procedure) nd ws therefore log-trnsformed prior to nlyses. All vlues presented re lest squre mens ± s.e.m. unless otherwise stted. RESULTS Tretment order (i.e. the order in which femles experienced the different diet regimes) ws initilly included s fixed effect in ll repeted mesures nlyses ut ws not found to e significnt term in ny of the models (P>0.1 for ll) nd ws therefore omitted. Body mss vrition nd mss independence of hemtologicl vriles Body mss vried cross the complete reproductive cycle for ll femles (N=29) on the high-qulity diet (F 4,23 =35.91, P<0.0001; Fig. 1A) s we hve previously descried (e.g. Slvnte nd Willims, 2002; Wgner et l., 2008). The etween-individul vrince component estimte ssocited with the model ws 0.61±0.22 (Z=2.77, P<0.003) nd the within-individul vrince component ws 0.49±0.08 (Z=6.28, P<0.0001). In reltion to diet qulity, there ws highly significnt diet reproductive stge interction for chnge in ody mss etween pre-reeding nd clutch completion, i.e. the pttern of chnge in ody mss vried y diet (F 2,53 =7.96, P<0.001; Fig.1C). The etween-individul vrince component estimte ssocited with the model ws 0.58±0.23 (Z=2.51, P<0.015) nd the within-individul vrince component ws 0.75±0.06 (Z=12.85, P<0.0001). On the high qulity diet, men ody mss incresed from pre-reeding to the stge, then decresed to levels significntly lower thn preceding stges t clutch completion (pre-reeding vs clutch completion, t 45 =4.36, P<0.008; vs clutch completion t 45 =8.92, P< (Fig. 1C). By contrst, there ws no significnt chnge in men ody mss cross reproductive stges on the low qulity diet (P>0.05 for ll comprisons; Fig. 1C). As previous studies hve found reltionship etween ody condition nd hemtocrit (reviewed y Fir et l., 2007), ody mss ws initilly included s covrite in ll repeted-mesures nlyses of hemtologicl vriles. However, ody mss did not significntly influence the vriles of interest (P>0.1 for ll) except in the nlysis of hemtocrit cross the complete reproductive cycle (P=0.002). Therefore, ody mss ws retined s covrite for nlysis of hemtocrit nd omitted from ll other nlyses of hemtologicl trits (hemogloin concentrtion, red lood cell numer, men cell volume, nd reticulocyte count). Vrition in hemtocrit Hemtocrit vried significntly with reeding stge (F 4,84 =13.28, P<0.0001; Fig. 1B), including mss s covrite in the model (F 1,104 =5.44, P=0.02). The etween-individul vrince component estimte ws 13.92±5.5 (Z=2.53, P=0.0026) nd the withinindividul vrince component ws 10.51±1.28 (Z=8.24, P<0.0001). Hemtocrit decresed significntly from pre-reeding to the stge (t 78 =6.99, P<0.0001), nd remined significntly lower thn pre-reeding levels t clutch completion (t 80 =4.4, P<0.0003; Fig. 1B). At htching, hemtocrit ws not significntly different from clutch completion or pre-reeding vlues (P>0.15 for oth) i.e. hemtocrit ppered to recover to seline levels (Fig. 1B). However, t fledging, hemtocrit decresed to levels significntly lower thn the preceding stge (htching vs fledging: t 80 =2.86, P<0.05) s well s pre-reeding seline levels (t 81 =4.42, P<0.0003; Fig.1B). For the diet-qulity experiment (N=23), there ws significnt diet reproductive stge interction for hemtocrit (F 2,60 =4.18, P<0.02; Fig. 1D). However, post-hoc pirwise nlyses did not detect ny significnt differences in hemtocrit t ny stge etween the different diets (P 0.2 for ll comprisons), nd chnge in hemtocrit etween reeding stges ws independent of diet qulity for ll pir- Body mss (g) A c,c,c C Fig. 1. Vrition in (A) ody mss nd (B) hemtocrit cross the reproductive cycle on high-qulity diet (different lowercse letters indicte significnt differences in mens t P<0.05 using Tukey Krmer djustment); nd vrition in (C) ody mss nd (D) hemtocrit cross the egg-lying cycle on highqulity (filled circles) versus low-qulity (open circles) diet. Vlues re lestsqure mens ± s.e.m Hemtocrit (%) B,c,c D 44 Pre-reeding Htch Fledging Pre-reeding

5 2964 E. C. Wgner, C. A. Stles nd T. D. Willims wise comprisons (P 0.10). After removing the interction term from the model, the min effects of diet qulity (F 1,22 =1.24, P=0.28) nd ody mss (F 1,100 =2.30, P=0.13) were not significnt, ut reproductive stge hd highly significnt effect on hemtocrit (F 2,38 =35.14, P<0.0001). The etween-individul vrince component estimte ssocited with the model ws 8.55±3.56 (Z=2.40, P<0.02) nd the within-individul vrince component ws 0.52±0.09 (Z=5.55, P<0.0001). Among femles on the high-qulity diet, men hemtocrit decresed significntly from pre-reeding irds to the stge (t 43 =5.12, P<0.0001), nd remined significntly lower thn pre-reeding vlues t clutch completion (t 52 =3.76, P<0.005; Fig.1D, Tle3). On the low-qulity diet, hemtocrit decresed significntly from pre-reeding to the stge (t 42 =7.05, P<0.0001), nd gin remined significntly lower thn pre-reeding vlues t clutch completion (t 46 =7.13, P<0.001; Fig.1D, Tle3). Vrition in plsm hemogloin concentrtion Plsm hemogloin concentrtion vried significntly with reeding stge (F 4,51 =4.85, P<0.02; Fig. 2A); the etweenindividul nd within-individul vrince component estimtes ssocited with the model were 1.86±0.78 (Z=2.38, P<0.01) nd 2.27±0.27 (Z=8.25, P<0.01), respectively. There were no significnt differences in hemogloin concentrtion etween the pre-reeding,, clutch completion or htching stges (P>0.1 for ll comprisons), ut hemogloin concentrtion t the fledging stge ws significntly lower thn ll other stges (P<0.05) except clutch completion (P=0.5; Fig. 2A). By contrst, for the diet-qulity nlysis, the diet reproductive stge interction ws not significnt (F 2,51 =0.45, P=0.64; Fig. 2C). After removing the interction term from the model, the min effect of diet qulity ws not significnt (F 1,32 =0.50, P=0.48), ut there ws significnt min effect of reproductive stge on vrition in hemogloin concentrtion (F 2,50 =13.44, P<0.0001). The etween-individul nd within-individul vrince component estimtes ssocited with the model were 0.33±0.74 (Z= 0.44, P=0.66) nd 0.44±0.10 (Z=4.44, P<0.0001) respectively. For the pooled diet groups, hemogloin concentrtion decresed from 16.19±0.35mgdl 1 in pre-reeding irds to 14.51±0.34 mg dl 1 t the stge (t 47 =4.49, P<0.0001), nd hemogloin concentrtion remined significntly lower thn pre-reeding levels t clutch completion (14.33±0.36mgdl 1 ; t 51 =4.69, P<0.0001). Vrition in red lood cell numer nd men cell volume Red lood cell (RBC) numer vried significntly with reeding stge (F 4,78 =11.85, P<0.001; Fig.2B). The etween-individul nd within-individul vrince component estimtes were 0.31±0.11 (Z=2.61, P<0.005) nd 0.28±0.05 (Z=6.17, P<0.0001) respectively. Men RBC decresed significntly from pre-reeding to the stge (t 76 =5.01, P<0.0001), nd remined significntly lower thn pre-reeding levels t clutch completion (t 78 =6.09, P<0.0001; Fig.2B). This ws followed y significnt increse in RBC from clutch completion to htching nd fledging stges (P<0.006 for oth comprisons); men RBC t htching nd fledging were not significntly different from pre-reeding vlues (P>0.3 for oth comprisons; Fig.2B). For the diet-qulity nlysis the diet reproductive stge interction ws not significnt (F 2,52 =0.85, P=0.44; Fig.2D). After the interction term ws removed from the model, the min effect of diet qulity ws not significnt (F 1,30 =0.01, P=0.9) ut reproductive stge did hve significnt effect on vrition in RBC numer (F 2,48 =18.03, P<0.001). The etweenindividul nd within-individul vrince component estimtes were 0.06±0.11 (Z=0.54, P=0.6) nd 0.51±0.09 (Z=5.65, P<0.0001), respectively. For pooled dt, pre-reeding RBC numer ( ± cellsμl 1 ) ws significntly higher thn RBC t the ( ± cellsμl 1 ; t 46 =5.49, P<0.0001) nd clutch completion stges ( ± cellsμl 1 ; t 50 =5.19, P<0.0001). Men red lood cell volume vried significntly with reeding stge (F 4,78 =3.13, P<0.02; Fig.3A). The etween-individul nd within-individul vrince component estimtes were ±58.97 (Z=2.27, P<0.025) nd ±42.06 (Z=7.93, P<0.0001), respectively. There were no significnt differences found in men cell volume mong pre-reeding, nd htching stges (P>0.05 for ll comprisons; Fig. 3A). However, men cell volume t clutch completion ws significntly greter thn pre-reeding men cell volume (t 77 = 2.74, P<0.05), wheres men cell volume t the fledging stge ws significntly lower thn men cell volume t clutch completion (t 82 =3.13, P<0.025; Fig.3A). For the comprison etween diets, the interction diet reproductive stge ws not significnt (F 2,48 =0.14, P=0.871; Fig.3C). After the interction term ws removed from the model, neither diet qulity (F 1,36 =0.38, P=0.54) nor reproductive stge (F 2,56 =1.20, P=0.31) hd significnt effect on vrition in red lood cell volume. The etween-individul nd within-individul vrince component estimtes were 25.57±52.99 (Z=0.48, P=0.63) nd 0.18±0.11 (Z=1.69, P=0.09), respectively. Vrition in reticulocyte counts Reticulocyte counts showed significnt vrition cross different stges of the reproductive cycle (F 4,40 =3.47, P<0.02; Fig.3B); the etween-individul nd within-individul vrince component estimtes were 1.32±1.94 (Z=0.68, P=0.25) nd 27.14±6.67 (Z=4.07, P<0.005), respectively. There ws no significnt difference found Tle 3. Reproductive output nd vrition in hemtocrit of femle zer finches in reltion to diet qulity Trit High-qulity diet Low-qulity diet Pre-reeding ody mss (g) 16.0± ±0.3 ody mss (g) 16.8± ±0.3 Men egg mss (g)*** 1.080± ±0.034 Clutch size (numer of eggs)*** 5.9± ±0.5 Clutch mss (g)*** 6.44± ±0.53 Lying intervl (dys) 7.1± ±0.6 Chnge in hemtocrit (%) Pre-reeding to 5.9±1.3 ( 8.8 to 3.1) 8.5±1.3 ( 11.2 to 5.8) One-egg stge to clutch completion +1.8±1.2 ( 0.9 to +4.4) +0.7±1.1 ( 1.8 to +3.2) Pre-reeding to clutch completion 4.2±1.5 ( 7.4 to 1.0) 7.8±1.5 ( 11.0 to 4.6) Vlues re lest-squred mens ± s.e.m. with 95% confidence intervls in prentheses; ***P<0.001 (N=23).

6 Hemtologicl chnges during egg lying 2965 [Hemogloin] (g dl 1 ) A, C Fig. 2. Vrition in (A) hemogloin concentrtion nd (B) red lood cell (RBC) numer cross the reproductive cycle on high-qulity diet (different lowercse letters indicte significnt differences in mens t P<0.05 using Tukey Krmer djustment); nd vrition in (C) hemogloin nd (D) red lood cell numer cross the egg-lying cycle on high-qulity (filled circles) versus low-qulity (open circles) diet. Vlues re lest-squre mens ± s.e.m. RBC numer (cells 10 6 µl 1 ) B D 3.6 Pre-reeding Htch Fledging Pre-reeding in reticulocyte counts etween pre-reeding,, clutch completion or fledging stges (P>0.05 for ll comprisons; Fig. 3B). However, the reticulocyte count t htching ws significntly greter thn t the pre-reeding (t 40 = 2.85, P<0.05) nd stge (t 40 = 3.31, P<0.02; Fig.3B). For the diet qulity experiment, the interction etween diet reproductive stge ws not significnt (F 2,27 =0.49, P=0.62; Fig.3D). After removing the interction term from the model, the min effect of diet qulity ws not significnt (F 1,18 =2.26, P=0.16), ut reproductive stge hd significnt effect on vrition in reticulocyte counts (F 2,27 =5.56, P<0.0085). The etween-individul nd within-individul vrince component estimtes were 34.52±10.83 (Z=3.19, P<0.001) nd 0.23±0.14 (Z= 1.64, P=0.10), respectively. On oth diets, the reticulocyte count t clutch completion (15.5±1.3%) ws significntly higher thn t the pre-reeding (10.3±1.3%; t 37 = 2.67, P<0.03) nd stges (9.4±1.2%; t 39 = 3.2, P<0.008). Effects of diet qulity on reproductive output On the high-qulity diet (ll femles, N=29), men egg size ws 1.106±0.021 g, men clutch size ws 5.9±0.5 eggs nd men clutch mss ws 6.444±0.497 g. For those femles tht htched chicks (N=18; 62% htching success), men rood size t htch ws 3.1±0.3 chicks, nd numer of chicks fledged ws 2.0±0.4. Correltionl nlyses showed no reltionship etween hemtologicl prmeters mesured t pre-reeding, or clutch completion stges nd men egg size, clutch size or rood size t htch nd fledging stges (P>0.05 in ll cses), nd no reltionship etween the chnge in hemtologicl prmeters from pre-reeding to the stge or from to clutch completion stge nd reproductive trits (P>0.05 in ll cses). Diet qulity significntly influenced reproductive output: femles on the high-qulity diet produced lrger clutches with lrger men egg mss (Tle 3). There ws no correltion etween reproductive output nd hemtologicl prmeters t ny stge or the chnge in these prmeters cross stges on either the high- or low-qulity diet (P>0.05 in ll cses). DISCUSSION We tested specific predictions generted from severl, non-mutully exclusive hypotheses for mechnisms underlying reproductive nemi ssocited with egg production (hemodilution, trnsient suppression of erythropoiesis, resource dependence) in reltion to (1) the time-course of development nd recovery from nemi, (2) chnges in specific hemtologicl trits, nd (3) the effect of diet qulity. Femle zer finches showed mrked decreses in hemtocrit (~6%), red lood cell counts (~8%), nd plsm hemogloin concentrtion (~9%) during egg production, even with high-qulity d liitum diet. It is likely tht the initil decrese in these trits involves hemodilution given the mrked chnges in plsm yolk precursor, clcium nd wter lnce t onset of lying (Morton, 1994; Reynolds nd Wldron, 1999; Slvnte nd Willims, 2002). However, our results provide strong support for the hypothesis tht erythropoiesis is trnsiently suppressed during egg-lying nd tht the recovery from nemi is reltively longlsting, extending through incution nd htching periods. Decresed hemtocrit, red lood cell counts nd hemogloin concentrtion did not recover t clutch completion, ut showed evidence of recovery to seline pre-reeding levels t htching, consistent with the estimted timescle for regenertive erythropoiesis following experimentlly induced nemi [7 14 dys (Domm nd Ter, 1946; Clrk et l., 1988)]. More importntly, there ws significnt time-dependent vrition in (1) the proportion of reticulocytes, which incresed t clutch completion ut peked t htching dys fter clutch completion, nd (2) in men red lood cell volume, which showed significnt increse t clutch completion. This is consistent with enhnced production nd relese of lrger immture cells into the circultion following suppression

7 2966 E. C. Wgner, C. A. Stles nd T. D. Willims Men cell volume (fl) A,,,c C Fig. 3. Vrition in (A) men cellulr volume nd (B) reticulocyte index (%) cross the reproductive cycle on highqulity diet (different lowercse letters indicte differences in mens t P<0.05 using Tukey Krmer djustment); nd vrition in (C) men cellulr volume nd (D) reticulocyte index cross the egglying cycle on high-qulity (filled circles) versus low-qulity (open circles) diet. Vlues re lest-squre mens ± s.e.m. Reticulocyte index (%) B,, D Pre-reeding Htch Fledging Pre-reeding of erythropoiesis. Finlly, we found no evidence tht development of nemi ssocited with egg production ws ffected y diet qulity, i.e. exogenous lipid nd protein resources ville to the lying femle. Of note, there were two key differences etween experiments regrding temporl vrition in hemtologicl trits. In the nlysis of temporl vrition cross the entire reproductive cycle, we found tht there ws no significnt difference in men hemogloin concentrtion nd reticulocyte counts etween pre-reeding, nd clutch completion stges, wheres in the diet qulity experiment, we found tht these trits vried significntly cross these stges. Inconsistencies etween the two nlyses cn e ttriuted to differences in smple size (i.e. not ll individuls were included in the diet qulity experiment) s well s the significnt degree of intrindividul vrition in hemtologicl trits typicl of this popultion (see Wgner et l., 2008). A reduction in hemtocrit during egg production hs een reported in numerous vin species (reviewed y Willims et l., 2004; Wgner et l., 2008), rnging from 1.5% in the gret tit (Hork et l., 1998) to 10% in the red-illed quele (Jones, 1983) reltive to pre-reeding levels. Furthermore, Klmch et l. (Klmch et l., 2004) concluded tht the extent of nemi ws proportionl to egg lying effort. The mgnitude of this chnge in hemtocrit is comprle to puttive dptive djustments of hemtocrit proposed to fcilitte oxygen uptke nd trnsfer during periods of intense metolic ctivity (reviewed y Sino et l., 1997; Sino et l., 1997). For exmple, incresed hemtocrit (rnge 1 20%) is ssocited with cclimtiztion to cold tempertures (Kuen et l., 1972; Crey nd Morton, 1976), low oxygen prtil pressures (Jeger nd McGrth, 1974; Keys et l., 1986; Prts et l., 1996), endurnce exercise or migrtion (Plomeque nd Plns, 1978; Soler et l., 1999; Lndys-Cinnelli et l., 2002) nd experimentlly elevted flight costs (Sino et l., 1997; Cuervo nd De Ayl, 2005). This supports tht chnges in hemtocrit ssocited with egg production (~6% decrese) re iologiclly relevnt nd could potentilly hve negtive impct on future reproduction vi effects on oxygen crrying cpcity nd eroic cpcity (Willims et l., 2004; Willims, 2005). Reticulocytosis, the enhnced relese of reticulocytes from the one mrrow due to stimultory effects of the hormone erythropoietin, is considered the est single indictor of intensified erythropoiesis in response to the tissue hypoxi ssocited with nemi (Fernndez nd Grindem, 2006). Although the proportions of reticulocytes present t the prereeding nd stges in our study were within the reference rnge reported for smll irds (~10%) (Cmpell nd Ellis, 2007), we noted n pproximte 5 7% increse in the proportion of reticulocytes during lter reeding stges (clutch completion nd htching) tht is consistent with the time-course of the reticulocyte response to nemi. Reticulocytosis is initilly oserved 2 4 dys fter induction of nemi (i.e. suppression of red lood cell production or lood loss), typiclly peks t 4 7dys, nd grdully declines within 2 3weeks of the originl insult (Fernndez nd Grindem, 2006). To our knowledge, our study is the first to exmine chnges in proportion of reticulocytes cross the reproductive cycle, lthough previous studies hve investigted chnges in the hemtopoietic orgns in this context (reviewed y Kendll, 1995; Klmch et l., 2004). For exmple, Jones (Jones, 1983) documented tht the thymus ecme enlrged nd ctively produced red lood cells in the redilled quele (Quele quele) during incution, nd suggested tht this functioned to ugment erythropoiesis in the one mrrow nd compenste for nemi during egg-lying. The chnges oserved in men red lood cell volume in the present study were lso consistent with the hypothesis tht erythropoiesis is suppressed during egg production. During initil stges of egg-lying, estrogen would inhiit the production of new red lood cells, ut existing

8 Hemtologicl chnges during egg lying 2967 red lood cells would mture nd splenic degrdtion of senescent erythrocytes would presumly continue (John, 1994). At clutch completion the decrese in plsm estrogen to non-reeding levels (Willims et l., 2004; Willims et l., 2005) would relese erythropoiesis from inhiition. This would result in higher proportion of reticulocytes in the plsm, which re lrger due to less condensed chromtin (Cmpell nd Ellis, 2007). In other words, fewer red lood cells would e present t clutch completion, ut greter proportion of these cells would e of lrger nd men cellulr volume would increse; gin, consistent with regenertive response to nemi (Fernndez nd Grindem, 2006). It is possile tht recovery from nemi, with enhnced nd sustined levels of erythropoiesis during incution, is regulted y high plsm prolctin levels tht re present during incution (Sockmn et l., 2006), s prolctin promotes erythropoiesis in mice (Jepson nd Lowenstein, 1964), nd hs een chrcterized s hemtopoietic growth fctor (Constntinescu et l., 1999; Welnik et l., 2001). In the present study, we found evidence for further reduction in hemtocrit nd hemogloin concentrtion t the end of the chickrering period (fledging), though t lower level thn tht seen during lying. In generl, reductions in hemtocrit during chick rering hve een ttriuted to decline in ody condition of provisioning prents nd/or relloction of resources to meet incresed energetic demnds of chick rering (Morton, 1994; Pp, 2002), lthough these correltive results contrst with the fct tht hemtocrit is positively correlted with experimentlly enlrged rood sizes in the gret tit (Hork et l., 1998). Although the energetic demnds of chick rering re presumly moderted in cptive species (e.g. sence of predtors, reduced forging costs, d liitum food), metolic rte is still significntly higher in cptivereeding zer finches during chick rering compred with prereeding or incuting irds (Vezin et l., 2006). However, n lternte explntion in our study is tht the decrese in hemtocrit t fledging my hve een due to second phse of estrogendependent nemi nd suppression of eythropoiesis in femles prepring to ly second clutch (i.e. re-lying). Dt from followup study (E.C.W. nd T.D.W., unpulished dt) support this: hemtocrit did not decrese linerly throughout the chick-rering period s would e expected if the energetic demnds of chick provisioning were negtively impcting mternl ody condition. Rther, hemtocrit remins t constnt level during chick rering, nd does not show decrese until the end of the fledging period, which is when femles would e inititing egg production for second clutch (E.C.W. nd T.D.W., unpulished dt). We predicted tht if reproductive nemi functioned to llow relloction of nutrients or energy to egg production, then hemtologicl chnges should e more mrked in femles red on protein- nd lipid-deficient diet, where the demnd for endogenous reserves would presumly e greter. Although there ws significnt interction etween diet qulity nd reproductive stge in the overll model for hemtocrit, we could not detect ny sttisticlly significnt differences in men hemtocrit in reltion to diet qulity within ny reeding stge, or in the chnge in hemtocrit etween reeding cross stges. In ddition, diet qulity hd no effect on hemogloin concentrtion or red lood cell numer lthough these trits lso decresed significntly during egg-lying. Therefore, we found little evidence to support tht diet qulity ffected the development or extent of nemi during egg production. To our knowledge, no other studies hve exmined whether diet qulity (e.g. protein nd lipid content) hs n effect on the degree of chnge in hemtologicl prmeters during egg production, lthough effects of diet restriction on hemtology hve een investigted in commercil species (Kuen et l., 1972; Grci et l., 1986; Mxwell et l., 1990). Food restriction is common prctice for improving iologicl nd economicl performnce in the domestic fowl, nd only severe food restriction progrms cuse hemtologicl prmeters to devite from the norml rnge (Mxwell et l., 1990). However, there is nother possile explntion for the pprent lck of effect of resource vilility on hemtologicl trits in our study: diet qulity did hve significnt effect on primry reproductive effort. Femles on the high-qulity diet produced igger clutches of lrger eggs, nd these eggs were likely of etter qulity (s indicted y the low htching success rte for eggs lid on the low-qulity diet). This suggests tht when diet qulity ws poor, egg-lying femles might hve trded-off reproduction for hemtologicl sttus, mintining hemtocrit, hemogloin nd red lood cell numer t some minimum functionl level t potentil cost of reduced reproductive investment. Our ody mss dt support this ide: lthough on the high-qulity diet femles were significntly hevier t the stge, there ws no difference in men ody mss t clutch completion etween trils, suggesting tht femles were not in poorer condition t cesstion of lying on the low-qulity diet. Mintennce of erythropoiesis t the cost of reduced reproductive output nd egg/offspring viility hs een demonstrted experimentlly in egglying Jpnese quil fed n iron-deficient diet (Grci et l., 1986). In comprison to control femles, when iron-deficient quil were provided with rdio-leled iron supplement, significntly higher mount of leled iron ws detected in the hemtopoietic orgns (i.e. one mrrow, liver, spleen) nd erythrocytes, ut no difference ws found in mount of leled iron found in the oviduct or eggs (Grci et l., 1986). In other words, lthough iron deposited in the yolk is essentil for emryo development nd postntl survivl, once iron ws provided to iron-deficient femles it ws preferentilly diverted to restoring red lood cell production nd hemtocrit levels in the lying femle herself nd not to reproductive output (Grci et l., 1986). In conclusion, our study demonstrtes tht trnsient suppression of erythropoiesis nd, susequently, incresed reticulocytosis re key components of reproductive nemi in egg-lying femles. Moreover, reticulocytosis, which is clssic physiologicl response ssocited with remission or recovery from nemi is prolonged in egg-lying femles (see lso Klmch et l., 2004) extending into incution nd chick rering. Thus, reproductive nemi could provide physiologicl sis for cost of egg production underlying the negtive crry-over effects of incresed egg production effort on susequent reproductive stges (i.e. incution nd chick rering) tht hve een widely reported in irds (e.g. Monghn et l., 1998; Nger et l., 2001). Future work should focus on linking these physiologicl chnges in hemtology to fitness, nd confirming tht this connectivity opertes vi effects on oxygen-crrying cpcity nd eroic cpcity, preferly using experimentl mnipultion of hemtologicl prmeters in egg-lying femles. LIST OF ABBREVIATIONS H hemogloin Hct hemtocrit MCV men cell volume RBC red lood cell count r Person correltion coefficient % Ret percent reticulocytes This study ws funded y Nturl Sciences nd Engineering Reserch Council of Cnd Discovery Grnt to T.D.W. We would like to thnk Julin Christins, Dvid Green, Chris Kennedy nd the T.D.W. l memers for useful suggestions

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