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Mi ochond ial unc ion is enhanced by hy oid ho mones du ing zeb a inch
de elopmen
© 2024 he Au ho s
Published e sion
Oe ele, Ma lene; Hau, Michaela; Ruuskanen, Su i; Casag ande, S e ania
Oe ele, M., Hau, M., Ruuskanen, S., & Casag ande, S. (2024). Mi ochond ial unc ion is
enhanced by hy oid ho mones du ing zeb a inch de elopmen . Royal Socie y Open Science,
11, A icle 240417. h ps://doi.o g/10.1098/ sos.240417
2024
Mi ochond ial unc ion is
enhanced by hy oid
ho mones du ing zeb a
inch de elopmen
Ma lene Oe ele1, Michaela Hau1,2, Su i Ruuskanen3 and
S e ania Casag ande1
1E olu iona y Physiology Resea ch G oup, Max Planck Ins i u e o Biological In elligence,
Ebe ha d-Gwinne -S asse, Seewiesen 82319, Ge many
2Depa men o Biology, Uni e si y o Kons anz, Kons anz D-78464, Ge many
3En i onmen al Physiology Resea ch G oup, Uni e si y o Jy äskylä, Seminaa inka u 15,
Uni e si y o Jy äskylä, Jy äskylä FI-40014, Finland
MO,0009-0008-4152-6320
An o ganism’s esponse o i s en i onmen is la gely
de e mined by changes in he ene gy supplied by ae obic
mi ochond ial me abolism ia adenosine iphospha e (ATP)
p oduc ion. ATP is especially impo an unde ene gy-
demanding condi ions, such as du ing apid g ow h. I
is cu en ly poo ly unde s ood how en i onmen al ac o s
in luence ene gy me abolism and mi ochond ial unc ioning,
bu ecen s udies sugges he ole o hy oid ho mones
(TH). TH a e key egula o s o g ow h and me abolism
and can be lexibly adjus ed o en i onmen al condi ions,
such as en i onmen al empe a u e o ood a ailabili y.
To es whe he TH enhancemen is causally linked o
mi ochond ial unc ion and g ow h, we p o ided TH o ally
a physiological concen a ions du ing he main g ow h
phase in zeb a inch (Taeniopygia gu a a) nes lings ea ed
in a challenging en i onmen . TH ea men accele a ed
maximal mi ochond ial wo king capaci y—a ai ha e lec s
mi ochond ial ATP p oduc ion, wi hou a ec ing g ow h. To
ou knowledge, his is he i s s udy o cha ac e ize he
egula ion o mi ochond ia by TH du ing de elopmen in
a semi-na u alis ic con ex and o add ess implica ions o
i ness- ela ed ai s, such as g ow h.
1. In oduc ion
O ganisms con inually adjus o hei su oundings, and ene gy
dynamics play a pi o al ole in shaping hese esponses. Cen al
© 2024 The Au ho (s). Published by he Royal Socie y unde he e ms o he C ea i e
Commons A ibu ion License h p://c ea i ecommons.o g/licenses/by/4.0/, which pe mi s
un es ic ed use, p o ided he o iginal au ho and sou ce a e c edi ed.
Resea ch
Ci e his a icle: Oe ele M, Hau M, Ruuskanen S,
Casag ande S. 2024 Mi ochond ial unc ion is
enhanced by hy oid ho mones du ing zeb a inch
de elopmen . R. Soc. Open Sci. 11: 240417.
h ps://doi.o g/10.1098/ sos.240417
Recei ed: 12 June 2023
Accep ed: 25 June 2024
Subjec Ca ego y:
O ganismal and e olu iona y biology
Subjec A eas:
e olu ion, ecology, cellula biology
Keywo ds:
mi ochond ia, hy oid ho mones, cellula
espi a ion, g ow h, me abolism
Au ho o co espondence:
Ma lene Oe ele
e-mail: [email p o ec ed].de
Elec onic supplemen a y ma e ial is a ailable
online a h ps://doi.o g/10.6084/
m9. igsha e.c.7349588.
Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024
o hese dynamics is ae obic mi ochond ial me abolism, which uels cellula unc ions h ough
he p oduc ion o adenosine iphospha e (ATP). The signi icance o ATP becomes pa icula ly
e iden unde ene gy-in ensi e condi ions, such as pe iods o apid g ow h. Howe e , he in e play
be ween en i onmen al ac o s, ene ge ic needs and mi ochond ial pe o mance emains insu icien ly
unde s ood. Recen esea ch has begun o shed ligh on his issue, indica ing ha hy oid ho mones
(TH) may se e as a link be ween en i onmen al in luences and me abolic adjus men [1,2]. TH plays a
cen al ole in e eb a e de elopmen , wi h a signi ican in luence on g ow h [1,3]. They a ec cellula
di e en ia ion, p oli e a ion [4] and me abolic egula ion [5], all o which a e essen ial o g ow h.
A he cellula le el, unde s anding he in e ac ion be ween TH and mi ochond ia is c ucial because
hei sec e ion is lexibly adjus ed o en i onmen al condi ions, such as clima ic condi ions o ood
a ailabili y [1,2,6–8]. Ae obic mi ochond ial me abolism—and hence ATP p oduc ion—de e mines an
o ganism’s ene gy me abolism [9] and hus he abili y o adjus o di e en ecological condi ions [10].
Mi ochond ia a e known o be impo an d i e s o li e-his o y decisions and adjus men s o ecological
challenges [11]. To da e, we know li le abou he mechanisms ha media e en i onmen al in luences
on mi ochond ial unc ion, bu i is plausible ha hey a e unde he in luence o TH [5,12–20].
Howe e , we a e s ill missing ecological pe spec i es o his ascina ing biochemical TH–mi ochond ia
in e play.
The con ol o mi ochond ial unc ion, hence o ene gy p oduc ion, is especially impo an du ing
g ow h and de elopmen , a c i ical li e-his o y s age cha ac e ized by high ene ge ic demands. G ow h
equi es p o ein syn hesis and cell eplica ion, which a e p ocesses ha necessi a e ene gy in he o m
o ATP. In e eb a es, op imal g ow h a e and la ge body size a e ela ed o su i al [21–24] and hus
ul ima ely de e mine i ness ou comes o pa en s and o sp ing. Ne e heless, only a ew s udies ha e
examined mi ochond ia and TH in an ecological con ex du ing o sp ing de elopmen and mos o
hem we e ca ied ou in bi ds. The de elopmen o bi ds, pa icula ly du ing he ea ly li e pe iod, is
cha ac e ized by apid g ow h and high me abolic demands [23,25]. A ian species exhibi ema kable
pheno ypic plas ici y and adap i e s a egies o cope wi h luc ua ing en i onmen al condi ions du ing
de elopmen , hus ep esen ing excellen models o in es iga e he e ec o change in TH on g ow h
and me abolism [6]. In g ea i (Pa us majo ) nes lings, a single TH-injec ion in o he eggs did no a ec
mi ochond ial espi a ion [26] o mi ochond ial densi y [27] a he ledgling s age. This could mean
ha TH ele a ion du ing he emb yonic phase does no ha e a las ing e ec , ha ing disappea ed by
he end o a ha chling’s de elopmen . Howe e , in al icial ha chlings, TH le els ha e been shown o
ise s eadily in he i s week a e ha ching and each adul le els a ound ledging—in pa allel wi h
he ha chlings’ g ow h pa e ns, and hus, pos -ha ching TH and g ow h pa e ns should be s udied
[25]. Al hough some s udies ha e add essed he e ec s o TH on g ow h in al icial nes lings, esul s
ac oss s udies a e inconclusi e [25–36]. In g ea i s, p ena al TH manipula ions had no e ec on
g ow h a any s age [26]. Howe e , in colla ed lyca che s (Ficedula albicollis), p ena al TH-ele a ions
lead o la ge chicks and ea lie ha ching, bu no long- e m consequences in he nes ling s age we e
obse ed [35]. Ne e heless, he en i onmen al ac o s ha a e known o p omo e ho monal lexibili y
a e mani old and i is s ill unknown whe he and how TH luc ua ions du ing he ledging s age
a ec mi ochond ia and g ow h. The e ec s o TH in he ledging s a e ha e mos ly been s udied in
poul y bu a concen a ions exceeding physiological anges. These s udies show ha bo h hypo- and
hype - hy oidal condi ions educe g ow h and de elopmen [6,37–39]. Fu he mo e, in ed-winged
blackbi d (Agelaius phoenicius) nes lings, hype - and hypo- hy oidal condi ions led o educed skele al
g ow h, compa ed wi h unmanipula ed nes lings [33] and in he zeb a inch (Taeniopygia gu a a),
TH-inhibi ion was ela ed o dec eased g ow h [34]. Howe e , i has emained unclea whe he hese
ela ionships a e induced by pha macological TH concen a ions o in ac do occu a physiological
TH concen a ions as well.
To s udy en i onmen ally induced TH esponses, i is c ucial o ope a e wi hin he physiological
ange o TH le els, which ep esen he e ol ed le els in esponse o en i onmen al signals. These
s udies a e i al o unde s anding he e ec s o en i onmen al challenges on li e-his o y ou comes.
Fo example, i is s ill no clea how an o ganism’s me abolism is adjus ed o changing en i onmen al
condi ions, like ising empe a u es o a lack o nu ien a ailabili y—bo h o which ha e been shown
o in luence TH-sec e ion [1,2,6,7,40]—and wha he consequences a e o i ness and li e-his o y
ou comes. G ow h is a aluable ai o s udy because g ow h pa e ns in ea ly li e can de e mine
su i al, especially in young passe ines [41].
Fu he mo e, i is impo an o analyse he me abolic consequences media ed by TH, because TH
in e ac wi h me abolism, and g ow h and me abolism pe se a e highly in e connec ed. Fo ins ance,
TH in luence g ow h ho mone sec e ion, a co e componen o g ow h egula ion [42]. G ow h ho mone
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can boos mi ochond ial e iciency, me abolic a e and biogenesis in andem wi h insulin-like g ow h
ac o o mee g ow h’s me abolic demands [43]. Thus, he e ec s o TH on mi ochond ia could be
indi ec , o ins ance ia g ow h ho mone. Howe e , TH has also been shown o a ec mi ochond ial
unc ion independen ly o g ow h in mul iple ways [44]. Fo example, by p omo ing he ansc ip ion
o mi ochond ial enzymes such as cy och ome a and c [15,19,45], ia he ansc ip ion o nuclea
genes, by in e ac ing wi h TH-binding si es in he DNA, ia cellula signalling cascades [15,46] o
di ec s imula ion o componen s o he mi ochond ial elec on anspo chain, such as complex
V [15,19]. Also, TH has been shown o s imula e mi ochond ial me abolic unc ion wi hin minu es
h ough mi ochond ial T3 binding si es o by inducing he ansc ip ion o mi ochond ial genes [46].
This p obably enables he o ganism o eac o sudden changes in ene ge ic demands. An example
o a sho - e m adjus men like his would be he need o boos he mo egula ion when expe iencing
a apid d op in en i onmen al empe a u e. Mi ochond ial he mogenesis in ol es leak espi a ion,
whe e ene gy is ans o med in o hea ins ead o ATP, and TH in e ac s wi h mi ochond ial uncou-
pling p o eins [19]— he p ima y d i e s o leak [47,48]. While TH is in ol ed in apid mi ochond ial
adjus men s such as o he mo egula ion, ano he impo an unc ion o TH is he adjus men o
mi ochond ial unc ion o sus ained physiological demands, such as g ow h o long-las ing en i on-
men al challenges [19]. These adjus men s can be con eyed h ough epigene ic p og amming by he
modi ica ion o me hyla ion pa e ns du ing de elopmen [49,50] o h ough p omo ing he ansc ip-
ion o mi ochond ial genes [46].
He e, we expe imen ally es ed he hypo hesis ha TH plays a ole in s imula ing mi ochond ial
unc ion du ing de elopmen , in addi ion o augmen ing g ow h. To be able o ans e ou indings
o na u al sys ems, we epea edly ele a ed ci cula ing le els o T3 ( iiodo hy onine, he biologically
ac i e hy oid ho mone) wi hin physiological anges du ing he mos apid pos -ha ch g ow h pe iod
in cap i e zeb a inches o add ess he ollowing ques ions: (i) do ele a ed TH concen a ions enhance
g ow h in zeb a inch nes lings? We expec ed TH- ea ed nes lings o ha e as e g ow h a es in bo h
skele al size and body mass. As a esul o accele a ed g ow h, TH- ea ed chicks may ei he ha e
la ge body sizes a he ime o ledging han con ol chicks, o al e na i ely, each he same inal size
as con ol chicks bu a an ea lie age. (ii) Is mi ochond ial unc ion enhanced by sus ained ele a ed
TH le els du ing g ow h? We expec ed TH o accele a e mi ochond ial espi a ion a e and oxida i e
phospho yla ion, which a e he p ima y de e minan s o ATP p oduc ion a e.
2. Me hods
2.1. Model species and housing
Zeb a inches om a local cap i e Seewiesen popula ion we e allowed o b eed o 23 weeks in
wo communal indoo a ia ies. The local cap i e zeb a inch popula ion was acqui ed in 2002 (231
indi iduals a he ime) om a colony held since 1985 by T. R. Bi khead a She ield Uni e si y, UK.
Fo mo e in o ma ion, see popula ion no. 18 in [51]. Each o he a ia ies was equipped wi h 30 wooden
nes boxes and 7−11 open nes ing cups, main ained a a cons an empe a u e o 21°C wi h humidi y a
59%, a a ligh : da k pho ope iod o 14 : 10 h. The bi ds we e p o ided wi h whi e co on and coconu
ib es as nes ing ma e ial, comme cial seed mix u e, supplemen a y comme cial egg ood, cu le ish
bone, g i and wa e ad libi um. The bi ds we e housed in communal a ia ies whe e access o seeds
and egg ood was cons ained by o e ing a 2 : 1 ood- o-husk a io o mimic na u ally challenging
ood esou ces [52]. Once pe week, he die was supplemen ed wi h salad and mul i i amins. The
husk–seed mix u e was p o ided in open boxes (60 × 40 × 12 cm) and he husk–egg ood mix u e in
la pla es. Food dishes we e placed on he loo . Nes s we e checked once pe day o new ha chlings,
and new ha chlings we e colou ma ked wi h ood colou ing on hei head o back down ea he s.
As pa o a sepa a e s udy, a 1 o 2 days pos -ha ch (dph), each chick was c oss- os e ed o c ea e
ei he la ge (six o se en chicks) o small b oods ( wo o h ee chicks). All chicks included in his s udy
we e ea ed in enla ged b oods and hus expe ienced a challenging en i onmen , which included a
possibili y o ood sho age, he need o enhanced begging a es and compe i ion o space [53]. The
mean b ood size du ing he expe imen o he o al du a ion was 5.56, s.d.: 0.699. This numbe is
sligh ly lowe han he ini ial b ood size o six o se en chicks, owing o na u ally occu ing dea h o
chicks h oughou he nes ling pe iod and asynch onous ledging.
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2.2. S udy design and TH adminis a ion
The hy oid gland p oduces mainly hy oxine (T4), which is con e ed o iiodo hy onine (T3)
by deiodinase enzymes [54] while main aining a ce ain T3/T4 a io in he plasma. Ou ea men
consis ed o T3, as i is he biologically ac i e o m o TH, because o i s gene ally highe ecep o
a ini y han T4 [55]. We aimed o ele a e T3 wi hin physiological anges du ing he main g ow h
phase; howe e , he daily T3 p oduc ion a e o ha chling zeb a inches is no known. Thus, we
conside ed he daily sec e ion a e o 1–3 µg T4 pe 100 g body mass as shown in o he bi d species
[56] in combina ion wi h da a on he T3 o T4 a io, which is a ound 0.5, as shown in he ci cula ion
o de eloping zeb a inches [57]. We selec ed a ea men dosage o 0.5 µg T3 pe 100 g body mass.
We achie ed his dosage by mul iplying he lowe ange o he known sec e ion a e o 1–3 µg T4 pe
100 g body mass wi h he known T3/T4 a io: (1 µg T4 pe 100 g body mass) × (0.5 T3/T4 a io). We
decided o use he lowe ange o he known T4 sec e ion a e o ou calcula ion o a oid o e dosing.
All con ol chicks ecei ed he ec o solu ion. Since mi ochond ial unc ion is sensi i e o s ess, o
example, media ed ia glucoco icoids [58], ou aim was o minimize handling s ess as much as
possible. Fo his eason, T3 was adminis e ed e e y second day (i.e. on dph 5, 7, 9, 11 and 13 wi h
a 200 µl pipe e, adjus ed o he exac body mass o each chick, ± 0.01 g) o ob ain peaks o ele a ed
T3 le els h oughou he main g ow h pe iod be ween 5 and 14 dph [33,59]. Blood sampling was
done a 14 dph (ca 20 h a e T3 adminis a ion in he expe imen al g oup) o measu e ci cula ing
T3 concen a ions and mi ochond ia unc ion. T3 was adminis e ed in a wi hin-b ood design, hence
wi hin each b ood, one andomly chosen chick ecei ed T3 (n = 19). Fo he con ol g oup, ei he
a sibling o he TH- ea ed chicks (ha ched in he same nes o o igin), was chosen o accoun o
po en ial gene ic simila i ies (sibling pai s be ween TH- ea ed and con ol chicks n = 16), o , because o
a lack o a ailabili y, chosen andomly ( o al chicks wi hou gene ic sibling n = 19). Fo an illus a ion
o he s udy design, see igu e 1. Each os e nes con ained a a ying numbe o ocal chicks ha
a e included in his s udy: 11 nes s con ained one o wo TH-chicks and one– h ee con ol; ou nes s
con ained one TH-chick each and no con ols; one nes con ained one con ol chick and no TH-chicks;
and wo nes s con ained h ee con ol chicks and no TH-chicks.
2.3. TH- alida ion g oup
A sepa a e g oup called ‘TH- alida ion’ (n = 7) was used o con i m ha he ea men led o he
desi ed peaks o ele a ed plasma T3 sho ly a e each adminis a ion. In he TH-chicks, he las T3
dose was gi en a noon on 13 dph and blood sampling (see §2.6) was done a 14 dph a a ound 09.00.
Hence, i is likely ha T3 gi en on 13 dph had been me abolized in la ge pa s be o e blood sampling
[33,59,60], and he e o e T3 plasma le els a ha ime poin do no e lec he ull ex en o which T3
plasma le els whe e aised on mul iple occasions h oughou he ea men pe iod. To con i m ha
he ea men did in ac aise T3 plasma le els as expec ed, we added he so-called ‘TH- alida ion’
g oup (n = 7) o ou expe imen . To ou knowledge, he o al up ake a e o T3 in bi ds has no been
es ed while in humans T3 plasma le els each hei peak abou 2.5 h a e o al adminis a ion [60].
Since bi ds ha e a as e me abolism han humans—e.g. mass-speci ic basal me abolic a es a e 30–40%
highe in bi ds han mammals [61]—we expec ed plasma T3 le els o each peak le els a ound 1 h pos
adminis a ion in ou nes lings. To con i m his, he TH- alida ion g oup ecei ed hei las ea men
dose on 14 dph, 1 h be o e aking he blood samples, ins ead o on 13 dph, like he es o he bi ds
in he s udy. O he han he inal dose lagging o 1 day (14 dph ins ead o 13 dph), he TH- alida ion
g oup was ea ed in he same way as he TH-chicks ( he expe imen al g oup). The TH- alida ion
g oup was used o con i m he T3- ea men and included in he g ow h cu e analysis, bu was no
pa o any o he analysis in his s udy.
To e i y ha he ea men ele a ed T3 plasma le els in he TH-chicks as expec ed, a sepa a e
eg ession model was un in which plasma T3-le els o he TH- alida ion g oup we e included in he
TH-chicks g oup, see §2.9. The TH- alida ion g oup was no conside ed o any o he analysis.
2.4. TH and ec o solu ion
Fo he p epa a ion o he ho mone solu ion, c ys alline T3 (3,3′,5- iiodo-L- hy onine, g ea e han 95%
HPLC, CAS numbe 6893-02-3, Sigma-Ald ich) was i s dissol ed in 0.1 M NaOH (NaOH eagen
g ade, g ea e han o equal o 98%, anhyd ous pelle s, CAS numbe 1310-7-3-2, dilu ed in MilliQ
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wa e ) and hen his T3–NaOH solu ion was dilu ed a 1 : 60 wi h ap wa e [62]. The concen a ion o
he solu ion was adjus ed, so ha a 1 µl inal solu ion could be adminis e ed pe g am body mass o
he chick [63], wi h a dosage o 0.5 µg T3 pe 100 g body mass. Fo he ec o p epa a ion, 0.1 M NaOH
solu ion was dilu ed a 1 : 60 wi h ap wa e . P epa ed ec o and ea men aliquo s we e s o ed a
−20°C and kep on ice un il use. Thawed aliquo s we e s o ed in he idge and used wi hin 1 day.
2.5. Chick g ow h
In o de o moni o he ha chlings’ de elopmen and o adjus he ea men dosage (see §2.2), body
mass (KERN, ± 0.01 o Pesola sp ing scale, ± 0.25 g) and a sus leng h we e aken a 0, 3, 5, 7, 9, 11,
13 and 14 dph. Because TH can a ec ei he skele al g ow h [33], muscula g ow h [64], a deposi ion
[65] o po en ially all o hese ac o s, we assessed body condi ion in addi ion o a sus leng h and
body mass measu es, o es i he e ec s o TH on ei he o hese g ow h pa ame e s a ec ed body
condi ion. To assess body condi ion, we used he scaled mass index (SMI) [66] be ween body mass
and a sus leng h a 5, 7, 9, 11, 13 and 14 dph. Pea son’s co ela ion coe icien o body mass and
a sus leng hs anged om 0.76 o 0.85, wi h p- alues less han 2.2 × 10−16. G ow h a es we e calcula ed
sepa a ely o a sus leng h and body mass and om he e on will be called ‘skele al g ow h a e’ and
‘mass g ow h a e’, espec i ely. G ow h a es we e calcula ed o 5 dph ( he s a o he ea men ), 7,
9, 11 and 13 dph in he ollowing way (see also [67]):
(Ta sus( )–Ta sus(s))/( –s),
whe e s is he p eceding measu emen o ime poin .
The ime poin 14 dph was no included in g ow h a e calcula ions. While all o he measu emen s
we e aken a noon, he measu emen a 14 dph was aken in he mo ning sho ly be o e blood
sampling. Thus, body mass was expec ed o be lowe a 14 dph compa ed wi h 13 dph because o he
ime o he day. Ta sus leng hs did no change be ween 13 and 14 dph.
16 sibling pai s,
19 chicks wi hou
gene ic sibling
Nes o O igin (n=23)
Fos e Nes (5-6chicks pe nes , n=18)
Con ol
n=25
Con ol
T3
TH-chicks
n=19
TH- ea ed chicks
n=26
TH-Valida ion
n=7
Figu e 1. Schema ic s udy design and sample sizes pe g oup. The assignmen o chicks o con ol (blue squa e), TH-chicks
(expe imen al g oup, o ange squa e) and TH- alida ion (no included in analysis) g oups is shown, as well as an example o he
wi hin-b ood design in he os e nes s. Pho o c edi : Kim Holzmann.
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2.6. Blood sampling
Blood samples we e aken wi hin 6 min o en e ing he a ia y by b achial enipunc u e om indi idu-
als a 5 dph, o a sepa a e s udy, and a 14 dph, o ob ain plasma samples and mi ochond ial measu es
o his s udy. A maximum amoun o 1% o he nes ling’s body mass was aken. Blood was s o ed
immedia ely on ice and cen i uged wi hin 2 h a 2000g o 10 min. Plasma was s o ed a −80°C and
analysed wi hin six mon hs. Red blood cells (RBCs) we e p ocessed immedia ely o mi ochond ial
measu emen s, acco ding o [68].
2.7. Mi ochond ial measu emen
A ian e y h ocy es possess unc ional mi ochond ia [69,70], allowing us o assess mi ochond ial
unc ion in in ac RBCs [68]. We used in ac RBCs o analyse mi ochond ial ai s in he na u al
physiological s a e o he bi d, wi hou a i icially al e ing hei immedia e en i onmen , e.g. subs a e
a ailabili y in he cell. We included he ollowing i e mi ochond ial a iables in his s udy: cellula
me abolic a e (CMR)— he o al amoun o oxygen consump ion in he cu en cellula condi ion;
p o on leakage (‘Leak’)— he po ion o mi ochond ial espi a ion no used o ATP p oduc ion;
oxida i e phospho yla ion (‘OxPhos’, calcula ed by sub ac ing leak espi a ion om CMR)— he
po ion o he mi ochond ial O2 consump ion cu en ly used o ATP p oduc ion; maximal wo king
capaci y o he elec on anspo sys em (‘ETS’)— he maximum capaci y o he mi ochond ia o
p oduce ATP unde he cu en cellula condi ions, including subs a e and elec on chain enzyme
a ailabili y; and he lux con ol a ios (FCR) L/CMR ( a io be ween leak and CMR) [68]. Fo assessing
mi ochond ial unc ion, a Cla k elec ode high- esolu ion espi ome e (Oxyg aph-2k, O obo os
Ins umen s, Innsb uck, Aus ia) was used on in ac RBCs. An aliquo o 7–30 µl RBC (mean 16.33 µl;
s.d.: 5.99 µl) was aken a e cen i uga ion o he blood sample om he bo om o he ube wi h a cu -
o pipe e ip. All measu emen s we e no malized o he olume o RBC used o he analysis. The
RBC aliquo was hen gen ly homogenized in cold espi a o y MiR05 bu e (0.5  mmol l−1 EGTA, 3
mmol l−1 MgCl2, 60  mmol l−1 po assium lac obiona e, 20  mmol l−1 au ine, 10  mmol l−1 KH2PO4,
20  mmol l−1 Hepes, 110  mmol l−1 suc ose, 15  mmol l−1 a y acid- ee bo ine se um albumin, pH 7.1).
The RBC-bu e solu ion was cen i uged a 500g o 5 min and he supe na an was emo ed. Washed
RBCs we e esuspended in esh Mi 05 bu e a 40°C and added o he espi ome e chambe . Then,
RBC oxygen consump ion was quan i ied acco ding o he ollowing p o ocol: (i) CMR, o en called
‘Rou ine’ espi a ion, is he basal espi a ion o he cell; (ii) 1  µg ml−1 oligomycin was added o he
chambe o inhibi ATP-dependen oxygen consump ion and o assess p o on leak, he e called ‘leak’
espi a ion; (iii) 1  µmol l−1, i a ion o ca bonyl cyanide m-chlo ophenyl hyd azine, a mi ochond ial
uncouple was added, o assess he maximal capaci y o he elec on anspo chain, so-called ‘ETS’;
(i ) 5  µmol l−1 an imycin a, o quan i y non-mi ochond ial oxygen consump ion. Non-mi ochond ial
oxygen consump ion was sub ac ed om all measu es abo e [68].
2.8. T3 ex ac ions—liquid ch oma og aphy–mass spec ome y
In b ie , 15 µl o plasma was mixed in me hanol and spiked wi h a known amoun o 13C12-T4
(La odan, Sweden) o ack eco e y. We i s pe o med liquid–liquid ex ac ion wi h chlo o o m
and me hanol. Samples hen wen h ough solid phase ex ac ion using chlo ide- o m anion exchange
esin (Bio-Rad) o pu i ica ion and we e d ied unde N2. The inal p oduc s we e e-dissol ed in
0.01% NH3 and plasma T3 and T4 we e simul aneously measu ed using a alida ed nano- low liquid
ch oma og aphy–mass spec ome y (LC-MS) p o ocol, o de ails see [71]. 13C6T3 and 13C6T4 ( om
Sigma) we e spiked in each sample as in e nal s anda ds, and T3 and T4 we e quan i ied using peak
a ea a ios o sample o i s in e nal s anda d. Se e al quali y con ol samples we e also included. Each
sample was analysed once, and ac oss sample ypes, he in e -assay a e age CV% was 6.9 o T3 and
4.4% o T4 [71]. MS da a we e acqui ed au oma ically using The mo Xcalibu so wa e (The mo Fishe
Scien i ic) and analysed using Skyline [72].
2.9. S a is ics
All s a is ical analyses we e done in R, e sion 4.2.2 [73]. We used gene alized linea models ia he
s an unc ion om he package ‘ s ana m’, e sion 2.21.3 [74] o calcula e pos e io means and 95%
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c edible in e als (C I), om 4000 simula ed alues om he join pos e io dis ibu ion o he model
pa ame e s. The p io dis ibu ion o s ana m can be classi ied as weakly in o ma i e, wi h a Gaussian
dis ibu ion and scaling based on he dis ibu ion o he da a. The de aul p io o each a iable is
a no mal dis ibu ion cen ed a 0. The scale (s.d.) o his p io is calcula ed by he p og am in a
da a-dependen way, wi h he scale ha is a unc ion o he esponse a iable (see elec onic supple-
men a y ma e ial, able S5, whe e p io s o each dependen a iable ha e been epo ed). P io s ha e
a mean o ze o o all g oup di e ences, hus a oiding bias owa ds signals om ei he he expe i-
men al o con ol g oup. The mean o he in e cep is pooled om da a om bo h g oups [75]. The
speci ic p io pa ame e s can be ound in elec onic supplemen a y ma e ial, able S5. We modelled he
esponse a iables pe p edic o s o in e es ia a Gaussian e o dis ibu ion and he ‘Iden i y’ link
unc ion. We epo s a is ical unce ain ies o he es ima es using 95% C I and pos e io p obabili ies
o speci ic hypo heses o each s a is ical es . Fu he , we calcula ed he pos e io p obabili ies (P), by
assessing he p opo ion o simula ed alues in line wi h he hypo hesis, o each model p edic o [76].
Residual dis ibu ion was assessed by using no mal quan ile–quan ile plo s, Tukey–Ascombe plo s
and by plo ing esiduals agains le e age. Model con e gence and i we e assessed ia he unc ion
‘shinys an’ in he ‘ s ana m’ package. Fu he mo e, he R2 was calcula ed, as well as he esidual
a iance o he model and he a iance o he andom ac o s.
To es o he e ec o he T3 ea men on ocal pa ame e s, he a iable ‘T ea men ’ (Con ol: n
= 25; TH-chicks: n = 19) was used as a p edic o . In all models, he nes o bi h ‘nes o o igin’ (n =
23) was included as a andom e ec , o con ol o gene ic simila i ies be ween siblings. To con ol
o di e en ea ing condi ions ac oss nes s, ‘ os e nes ’ (n = 18) was also included as a andom
ac o in all models. To accoun o possible ime e ec s (23 weeks o he expe imen ), we included
sampling o de ep esen ing he o e all ha ching o de as a co a ia e in all models. Sampling o de
is a con inuous sequence o he o de o bi h o indi iduals, simila o Julian da es. All con inuous
p edic o a iables we e s anda dized by compu ing z-sco es ((x-mean)/s.d.). Fo some indi iduals,
da a we e missing (e.g. low amoun o blood, ailed mi ochond ial measu emen s, missing da a), hence
sample sizes a e sligh ly di e en be ween he models (see elec onic supplemen a y ma e ial, ables
S2–S5). Plo s we e gene a ed using he package ‘ggplo 2’ [77].
In o de o assess whe he he T3 plasma le els di e ed be ween he g oups, and whe he his
di e ence was enhanced by he TH- alida ion g oup (see §2.3), we an one model in which he
TH- alida ion g oup was included in he TH-chicks g oup (Con ol: n = 25; TH-chicks including
TH- alida ion g oup: n = 26) and one model wi hou he TH- alida ion g oup (Con ol: n = 25;
TH-chicks wi hou he TH- alida ion g oup: n = 19). T3 alues we e log- ans o med o achie e a
no mal dis ibu ion o he da a and o ob ain he bes i o he models.
F equen is esul s can be ound in elec onic supplemen a y ma e ial, ables S6–S8.2. I one o he
andom ac o s (1|nes o o igin) o (1| os e nes ) explained close o 0 a iance in he equen is
models, i was emo ed om he model o a oid con e gence issues. Resul s om equen is models
a e he same compa ed wi h Bayesian in e e ence ac oss all models.
2.10. Mo phome ics
To es he hypo hesis ha he TH- ea men posi i ely a ec ed he a o emen ioned mo phological
measu es, we an one model wi h a sus leng h a 14 dph, one wi h body mass a 14 dph and one
wi h SMI a 14 dph. We i s es ed o a di e ence in baseline measu es a 5 dph in all h ee a iables,
by unning one model pe a iable wi h he baseline measu e a 5 dph as an ou come a iable. As
sampling o de did no ha e a meaning ul e ec in he model o a sus leng h a 5 dph, we emo ed
i because o con e gence issues. Ta sus leng h a 5 dph, hence be o e he onse o he ea men ,
was smalle in TH-chicks (es ima e [95% C I], P(ß < 0): −0.59 [−1.028; −0.163], 0.995. See elec onic
supplemen a y ma e ial, able S3, igu e S1). The e was no di e ence be ween he g oups in body mass
a 5 dph and SMI a 5 dph (see elec onic supplemen a y ma e ial, able S3 and igu es S2 and S3). We
con olled o baseline measu es a 5 dph in all h ee models es ing he pa ame e s a 14 dph ( a sus
leng h, body mass, SMI), o accoun o he ela i ely high bu na u al a ia ion a 5 dph, which can be
seen in he dispe sion o he aw da a (elec onic supplemen a y ma e ial, igu es S1–S3). In his way,
we could disen angle he e ec s o he ea men om exis ing p e- ea men di e ences.
To es o di e ences in g ow h be ween he g oups, we used gene alized addi i e mixed models
(GAMMs) wi h he s ana m unc ion s an_gamm4 (see [67]). We included ‘ ea men ’ as a ixed e ec
and a smoo hing e m wi h an in e ac ion be ween age and ea men , o allow o di e en shapes o
he cu es in he wo g oups. Fu he , we included andom in e cep s o ID o e Age o accoun o
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di e ences in indi idual g ow h ajec o ies. GAMMs we e calcula ed o skele al and mass g ow h
a es, as well as o body mass (g), a sus (mm) and SMI o he ime poin s 5 dph (s a o he
ea men ), 7, 9, 11 and 13 dph.
2.11. Mi ochond ia
To es o di e ences in he i e mi ochond ial ai s o in e es (CMR, OxPhos, ETS, Leak and FCR
L/CMR) we an one model pe ai , wi h he ai being he esponse a iable. CMR, OxPhos, ETS
and Leak we e log- ans o med o achie e a no mal dis ibu ion o he da a and o ob ain he bes
i o he models. Du ing he ea men , we adminis e ed T3 e e y second day o ob ain peaks o T3
plasma le els, wi h he aim o egula ly ele a ing T3 h oughou he main g ow h phase. In humans,
TH ha e been shown o a ec mi ochond ia di e en ly in he sho e m (wi hin minu es o hou s a e
ea men ) e sus he long e m (wi hin weeks) [15]. Ou s udy ques ion aimed o add ess he e ec s
o ele a ed T3 on mi ochond ia h oughou he main g ow h phase. Acu e le els o T3 a he ime o
sampling may di e be ween indi iduals, o example owing o a ying speeds o me abolizing he
T3 o he eeding s a us a he ime o adminis a ion. Thus, we included acu e T3 plasma le els as
a co a ia e o con ol o immedia e e ec s o T3 on mi ochond ial unc ion ha may di e om he
e ec s o p olonged ele a ed T3 le els h oughou he ea men pe iod. Fu he mo e, we included
body mass a 14 dph (which was no s a is ically meaning ully a ec ed by he ea men , see elec onic
supplemen a y ma e ial, able S3) as a co a ia e, o con ol o body mass di e ences no a ibu ed o
he ea men . We e- an he models o mi ochond ial ai s wi hou acu e T3 le els o es whe he
he esul s changed, and we did no ind a di e ence in ei he model i o meaning ulness o esponse
a iables (see elec onic supplemen a y ma e ial, able S4.1). Ta sus leng h be o e he onse o he
ea men (a 5 dph) happened o be smalle in TH-chicks han in con ol chicks, while he g oups
did no di e in a sus leng h a 14 dph. To con i m ha mi ochond ial ai s we e no a ec ed by
po en ial ca ch-up g ow h, we e- an he models o mi ochond ial ai s wi h a sus leng h a 5 dph
as a co a ia e. Body mass a 14 dph was hen emo ed om he models since body mass a 14 dph
is expec edly highly dependen on a sus leng h a 5 dph. Ta sus leng h a 5 dph did no ha e a
meaning ul e ec on mi ochond ial ai s, no did model i o meaning ulness o ou come a iables
change (see elec onic supplemen a y ma e ial, able S4.2 o ull ou pu s o models including a sus
leng h a 5 dph).
3. Resul s
3.1. TH- ea men
As expec ed, T3 concen a ions 1 h a e adminis a ion in he sepa a e alida ion g oup ‘TH- alida-
ion’ (pmol ml−1 mean: 2.568, s.e.: 0.313), and in he expe imen al g oup abou 24 h a e adminis a ion
(pmol ml−1 mean: 1.825, s.e.: 0.067) we e inc eased and wi hin he ange o he physiological le els
ound in he con ol g oup (pmol ml−1 mean: 1.68, s.e.: 0.991). T3 plasma le els in he TH-chicks anged
om 1.315 o 2.25 pmol ml−1, which is wi hin he ange o 0.825 o 2.674 pmol ml−1 o he con ol
g oup. As expec ed, T3 le els in he TH-chicks we e s a is ically ma ginally highe han in he con ol
g oup (pmol ml−1 es ima e [95% C I], P(ß > 0): 0.127 [−0.016; 0.268], 0.962) and meaning ully highe
when TH- alida ion chicks and TH-chicks we e analysed oge he (pmol ml−1 es ima e [95% C I], P(ß >
0): 0.206 [0.066; 0.348], 0.997) (see igu e 2, elec onic supplemen a y ma e ial, able S2). Fu he mo e,
T3-le els we e simila o hose obse ed in nes lings o o he passe ine species, ed-winged blackbi ds
(Agelaius phoenicius) [33] and g ea i s [78].
3.2. G ow h
TH-chicks had smalle a sus leng hs a 5 dph be o e he onse o he ea men (es ima e [95% C I], P(ß
< 0): −0.59 [−1.028; −0.163], 0.995; see elec onic supplemen a y ma e ial, able S3, igu e S1). The las
ea men dose was gi en on 13 dph and 1 day a e he las ea men dose, on 14 dph, he TH-chicks
did no di e in body size (in a sus leng h o body mass) compa ed wi h he con ol g oup ( a sus
leng h in mm es ima e [95% C I], P(ß < 0): −0.006 [−0.327; 0.317], 0.514). Body mass in g ams es ima e
[95% C I], P(ß < 0): −0.381 [−0.97; 0.21], 0.906; see elec onic supplemen a y ma e ial, igu es S4 and S5).
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Re e ences
1. Beh inge V, Deimel C, Hohmann G, Neg ey J, Schaebs FS, Deschne T. 2018 Applica ions o non-in asi e hy oid ho mone measu emen s in
mammalian dcology, g ow h, and main enance. Ho m. Beha . 105, 66–85. (doi:10.1016/j.yhbeh.2018.07.011)
2. Kees a S, Högq is Tabo V, Al e gne A. 2021 Rein e p e ing pa e ns o a ia ion in human hy oid unc ion: an e olu iona y ecology
pe spec i e. E ol. Med. Public Heal h 9, 93–112. (doi:10.1093/emph/eoaa043)
3. McNabb FMA. 2007 The hypo halamic-pi ui a y- hy oid (HPT) axis in bi ds and i s ole in bi d de elopmen and ep oduc ion. C i . Re . Toxicol.
37, 163–193. (doi:10.1080/10408440601123552)
4. Pascual A, A anda A. 2013 Thy oid ho mone ecep o s, cell g ow h and di e en ia ion. Biochim. e Biophys. Ac a 1830, 3908–3916. (doi:10.
1016/j.bbagen.2012.03.012)
5. Mullu R, Liu YY, B en GA. 2014 Thy oid ho mone egula ion o me abolism. Physiol. Re . 94, 355–382. (doi:10.1152/phys e .00030.2013)
6. McNabb FMA. 2006 A ian hy oid de elopmen and adap i e plas ici y. Gen. Comp. Endoc inol. 147, 93–101. (doi:10.1016/j.ygcen.2005.12.
011)
7. Klein A, Kulcsá M, K ízsik V, Má ics R, Rudas P, Tö ök J, Huszenicza G. 2006 E ec s o en i onmen al empe a u e on hy oid ho mones in he
ba n owl (Ty o alba). Ac a Ve . Hung. 54, 321–331. (doi:10.1556/AVe .54.2006.3.3)
8. DuRan SE, Ca e AW, Den e RJ, Hepp GR, Hopkins WA. 2014 A e hy oid ho mones media o s o incuba ion empe a u e-induced pheno ypes
in bi ds. Biol. Le . 10, 20130950. (doi:10.1098/ sbl.2013.0950)
9. Delhaye J, Salamin N, Roulin A, C iscuolo F, Bize P, Ch is e P. 2016 In e speci ic co ela ion be ween ed blood cell mi ochond ial ROS
p oduc ion, ca diolipin con en and longe i y in bi ds. Age 38, 433–443. (doi:10.1007/s11357-016-9940-z)
10. B own JH, Gillooly JF, Allen AP, Sa age VM, Wes GB. 2004 Towa d a me abolic heo y o ecology. Ecology 85, 1771–1789. (doi:10.1890/03-
9000)
11. Koch RE e al. 2021 In eg a ing mi ochond ial ae obic me abolism in o ecology and e olu ion. T ends Ecol. E ol. 36, 321–332. (doi:10.1016/j.
ee.2020.12.006)
12. Hulbe AJ. 2000 Thy oid ho mones and hei e ec s: a new pe spec i e. Biol. Re . Camb. Philos. Soc. 75, 519–631. (doi:10.1017/
s146479310000556x)
13. B en GA. 2012 Mechanisms o hy oid ho mone ac ion. J. Clin. In es . 122, 3035–3043. (doi:10.1172/JCI60047)
14. Sil es i E, Schia o L, Lomba di A, Goglia F. 2005 Thy oid ho mones as molecula de e minan s o he mogenesis. Ac a Physiol. Scand. 184,
265–283. (doi:10.1111/j.1365-201X.2005.01463.x)
15. Cio i F, Senese R, Lanni A, Goglia F. 2013 Thy oid ho mones and mi ochond ia: wi h a b ie look a de i a i es and analogues. Mol. Cell.
Endoc inol. 379, 51–61. (doi:10.1016/j.mce.2013.06.006)
16. Liu JS, Chen YQ, Li M. 2006 Thy oid ho mones inc ease li e and muscle he mogenic capaci y in he li le bun ings (Embe iza pusilla). J. The m.
Biol. 31, 386–393. (doi:10.1016/j.j he bio.2006.01.002)
17. Noli L e al. 2020 E ec s o hy oid ho mone on mi ochond ia and me abolism o human p eimplan a ion emb yos. S em Cells 38, 369–381.
(doi:10.1002/s em.3129)
18. W u niak-Cabello C, Casas F, Cabello G. 2001 Thy oid ho mone ac ion in mi ochond ia. J. Mol. Endoc inol. 26, 67–77. (doi:10.1677/jme.0.
0260067)
19. Cio i F, Giacco A, Goglia F, Sil es i E. 2022 Bioene ge ic aspec s o mi ochond ial ac ions o hy oid ho mones. Cells 11, 997. (doi:10.3390/
cells11060997)
20. Van I allie CM. 1990 Thy oid ho mone and dexame hasone inc ease he le els o a messenge ibonucleic acid o a mi ochond ially encoded
subuni bu no o a nuclea -encoded subuni o cy och ome c oxidase. Endoc inology 127, 55–62. (doi:10.1210/endo-127-1-55)
21. Cha no EL. 2004 The op imal balance be ween g ow h a e and su i al in mammals. E ol. Ecol. Res. 6, 307–313.
22. Fo sman A. 1993 Su i al in ela ion o body size and g ow h a e in he adde , Vipe a be us. J. Anim. Ecol. 62, 647. (doi:10.2307/5385)
23. Rickle s RE. 1984 The op imiza ion o g ow h a e in al icial bi ds. Ecology 65, 1602–1616. (doi:10.2307/1939139)
24. Pla d F, Gailla d JM, Coulson T, Hewison AJM, Douha d M, Klein F, Delo me D, Wa nan C, Bonen an C. 2015 The in luence o bi h da e ia
body mass on indi idual i ness in a long-li ed mammal. Ecology 96, 1516–1528. (doi:10.1890/14-0106.1)
25. McNabb FMA, S an on FW, Dicken SG. 1984 Pos -ha ching hy oid de elopmen and body g ow h in p ecocial s al icial bi ds. Comp. Biochem.
Physiol. A Physiol. 78, 629–635. (doi:10.1016/0300-9629(84)90609-1)
26. Cossin-Se in N, Hsu BY, Ma ciau C, Viblanc VA, Ruuskanen S, S ie A. 2022 E ec o p ena al glucoco icoids and hy oid ho mones on
de elopmen al plas ici y o mi ochond ial ae obic me abolism, g ow h and su i al: an expe imen al es in wild g ea i s. J. Exp. Biol. 225,
jeb243414. (doi:10.1242/jeb.243414)
27. Hsu BY, Cossin-Se in N, S ie A, Ruuskanen S. 2023 P ena al hy oid ho mones accele a e pos na al g ow h and elome e sho ening in wild
g ea i s. J. Exp. Biol. 226, jeb243875. (doi:10.1242/jeb.243875)
28. Sa aude T, Hsu BY, G oo huis TGG, Ruuskanen S. 2020 Manipula ion o p ena al hy oid ho mones does no a ec g ow h o physiology in
nes ling pied lyca che s. Physiol. Biochem. Zool. 93, 255–266. (doi:10.1086/709030)
29. Hsu BY, Dijks a C, Da as VM, de V ies B, G oo huis TGG. 2017 Ma e nal hy oid ho mones enhance ha ching success bu dec ease nes ling body
mass in he ock pigeon (Columba li ia). Gen. Comp. Endoc inol. 240, 174–181. (doi:10.1016/j.ygcen.2016.10.011)
15
oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417
Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024

30. Hsu BY, Sa aude T, Cossin-Se in N, C ombecque M, S ie A, Ruuskanen S. 2020 Tes ing o con ex -dependen e ec s o p ena al hy oid
ho mones on o sp ing su i al and physiology: an expe imen al empe a u e manipula ion. Sci. Rep. 10, 14563. (doi:10.1038/s41598-020-
71511-y)
31. Ruuskanen S, Da as VM, Visse ME, G oo huis TGG. 2016 E ec s o expe imen ally manipula ed yolk hy oid ho mone le els on o sp ing
de elopmen in a wild bi d species. Ho m. Beha . 81, 38–44. (doi:10.1016/j.yhbeh.2016.03.006)
32. S ie A, Hsu BY, Ma ciau C, Doligez B, Gus a sson L, Bize P, Ruuskanen S. 2020 Bo n o be young? P ena al hy oid ho mones inc ease ea ly-li e
elome e leng h in wild colla ed lyca che s. Biol. Le . 16, 20200364. (doi:10.1098/ sbl.2020.0364)
33. Si sa TS, Si sa SKG, P ice ER, Pineda M, Dzialowski EM. 2022 Manipula ing plasma hy oid ho mone le els al e s de elopmen o endo he my
and en ila ion in nes ling ed-winged blackbi ds. F on . Physiol. 13, 1027257. (doi:10.3389/ phys.2022.1027257)
34. Rainwa e TR, Wood MB, Millam JR, Hoope MJ. 2008 E ec s o pe chlo a e on g ow h and beha io o a g ani o ous passe ine, he zeb a inch
(Taeniopygia gu a a). A ch. En i on. Con am. Toxicol. 54, 516–524. (doi:10.1007/s00244-007-9045-x)
35. Hsu BY, Doligez B, Gus a sson L, Ruuskanen S. 2019 T ansien g ow h-enhancing e ec s o ele a ed ma e nal hy oid ho mones a no appa en
oxida i e cos du ing ea ly pos na al pe iod. J. A ian Biol. 50. (doi:10.1111/ja .01919)
36. S ie A, Hsu BY, Cossin-Se in N, Ga cin N, Ruuskanen S. 2021 F om clima e wa ming o accele a ed cellula ageing: an expe imen al s udy in
wild bi ds. Physiology. (doi:10.1101/2021.12.21.473625)
37. King DB, May JD. 1984 Thy oidal in luence on body g ow h. J. Exp. Zool. 232, 453–460. (doi:10.1002/jez.1402320311)
38. Decuype e E, Van As P, Van de Gey en S, Da as VM. 2005 Thy oid ho mone a ailabili y and ac i i y in a ian species: a e iew. Domes . Anim.
Endoc inol. 29, 63–77. (doi:10.1016/j.domaniend.2005.02.028)
39. Jianhua H, Oh suka A, Hayashi K. 2000 Selenium in luences g ow h ia hy oid ho mone s a us in b oile chickens. B . J. Nu . 84, 727–732. (doi:
10.1017/S0007114500002087)
40. Rudas P, Pe hes G. 1984 The impo ance o he pe iphe al hy oid ho mone deiodina ion in adap a ion o ambien empe a u e in he chicken
(Gallus domes icus). Comp. Biochem. Physiol. A Physiol. 77, 567–571. (doi:10.1016/0300-9629(84)90230-5)
41. Me ill L, Jones TM, B awn JD, Wa d MP. 2021 Ea ly‐Li e pa e ns o g ow h a e linked o le els o pheno ypic ai co a iance and pos ledging
mo ali y ac oss a ian species. Ecol. E ol. 11, 15695–15707. (doi:10.1002/ece3.8231)
42. Mu ay PG, Higham CE, Clay on PE. 2015 60 yea s o neu oendoc inology: he hypo halamo-GH axis: he pas 60 yea s. J. Endoc inol. 226,
T123–40. (doi:10.1530/JOE-15-0120)
43. Hu B, Li H, Zhang X. 2021 A balanced ac : he e ec s o GH–GHR–Ig 1 axis on mi ochond ial unc ion. F on . Cell De . Biol. 9, 630248. (doi:10.
3389/ cell.2021.630248)
44. Ha pe ME, Sei e EL. 2008 Thy oid ho mone e ec s on mi ochond ial ene ge ics. Thy oid 18, 145–156. (doi:10.1089/ hy.2007.0250)
45. Van I allie CM. 1990 Thy oid ho mone and dexame hosone inc ease he le el o a mRNA o mi ochond ially encoded subuni bu no o a
nuclea -encoded subuni o cy och ome c oxidase. Endoc inology 127, 55–62. (doi:10.1210/endo-127-1-55)
46. Lanni A, Mo eno M, Goglia F. 2016 Mi ochond ial ac ions o hy oid ho mone. Comp . Physiol. 1591–1607. (doi:10.1002/cphy.c150019)
47. Nicholls DG. 2021 Mi ochond ial p o on leaks and uncoupling p o eins. Biochim. Biophys. Ac a - Bioene g. 1862, 148428. (doi:10.1016/j.bbabio.
2021.148428)
48. Bouillaud F, Al es-Gue a MC, Ricquie D. 2016 UCPs, a he in e ace be ween bioene ge ics and me abolism. Biochim. Biophys. Ac a - Mol. Cell
Res. 1863, 2443–2456. (doi:10.1016/j.bbamc .2016.04.013)
49. Fonseca TL, Ga cia T, Fe nandes GW, Nai TM, Bianco AC. 2021 Neona al hy oxine ac i a ion modi ies epigene ic p og amming o he li e . Na .
Commun. 12, 4446. (doi:10.1038/s41467-021-24748-8)
50. Anselmo J, Cha es CM. 2020 Physiologic signi icance o epigene ic egula ion o hy oid ho mone a ge gene exp ession. Eu . Thy oid J. 9, 114–
123. (doi:10.1159/000506423)
51. Fo s meie W, Segelbache G, Muelle JC, Kempenae s B. 2007 Gene ic a ia ion and di e en ia ion in cap i e and wild zeb a inches
(Taeniopygia gu a a). Mol. Ecol. 16, 4039–4050. (doi:10.1111/j.1365-294X.2007.03444.x)
52. Lemon WC. 1991 Fi ness consequences o o aging beha iou in he zeb a inch. Na u e 352, 153–155. (doi:10.1038/352153a0)
53. De Kogel CH. 1997 Long- e m e ec s o b ood size manipula ion on mo phological de elopmen and sex-speci ic mo ali y o o sp ing. J.
Anim. Ecol. 66, 167. (doi:10.2307/6019)
54. Da as VM, Van He ck SLJ. 2012 Iodo hy onine deiodinase s uc u e and unc ion: om ascidians o humans. J. Endoc inol. 215, 189–206. (doi:
10.1530/JOE-12-0204)
55. Da as VM, Houb ech s AM, Van He ck SLJ. 2015 In acellula hy oid ho mone me abolism as a local egula o o nuclea hy oid ho mone
ecep o -media ed impac on e eb a e de elopmen . Biochim. Biophys. Ac a - Gene Regul. Mech. 1849, 130–141. (doi:10.1016/j.bbag m.
2014.05.004)
56. McNabb FMA, Da as VM. 2015 Chap e 24 - Thy oids. In S u kie’s a ian physiology (ed. CG Scanes), pp. 535–547, 6 h edn. San Diego, CA:
Academic P ess. (doi:10.1016/B978-0-12-407160-5.00024-5)
57. Yamaguchi S, Hayase S, Aoki N, Takeha a A, Ishigohoka J, Ma sushima T, Wada K, Homma KJ. 2017 Sex di e ences in b ain hy oid ho mone
le els du ing ea ly pos -ha ching de elopmen in zeb a inch (Taeniopygia gu a a). PLoS One 12, e0169643. (doi:10.1371/jou nal.pone.
0169643)
58. Casag ande S, S ie A, Monaghan P, Lo eland JL, Bone W, Lupi S, T e isi R, Hau M. 2020 Inc eased glucoco icoid concen a ions in ea ly li e
cause mi ochond ial ine iciency and sho elome es. J. Exp. Biol. 223, jeb222513. (doi:10.1242/jeb.222513)
16
oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417
Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024
59. Wilson CM, McNabb FMA. 1997 Ma e nal hy oid ho mones in Japanese quail eggs and hei in luence on emb yonic de elopmen . Gen. Comp.
Endoc inol. 107, 153–165. (doi:10.1006/gcen.1997.6906)
60. Jonklaas J, Bu man KD, Wang H, La ham KR. 2015 Single-dose T3 adminis a ion: kine ics and e ec s on biochemical and physiological
pa ame e s. The . D ug Moni . 37, 110–118. (doi:10.1097/FTD.0000000000000113)
61. Ga ilo VM, Golube a TB, Wa ack G, Bushue AV. 2022 Me abolic scaling in bi ds and mammals: how axon di e gence ime, phylogeny, and
me abolic a e a ec he ela ionship be ween scaling exponen s and in e cep s. Biology 11, 1067. (doi:10.3390/biology11071067)
62. Van He ck SLJ, Geysens S, Delbae e J, Tylzanowski P, Da as VM. 2012 Exp ession p o ile and hy oid ho mone esponsi eness o anspo e s
and deiodinases in ea ly emb yonic chicken b ain de elopmen . Mol. Cell. Endoc inol. 349, 289–297. (doi:10.1016/j.mce.2011.11.012)
63. Roches e JR, Heiblum R, Rozenboim I, Millam JR. 2008 Pos -ha ch o al es ogen exposu e educes o iduc and egg mass and al e s nes -
building beha io in adul zeb a inches (Taeniopygia gu a a). Physiol. Beha . 95, 370–380. (doi:10.1016/j.physbeh.2008.07.008)
64. Dea on KE, Bishop CM, Bu le PJ. 1998 Tissue-speci ic e ec s o hypo hy oidism on pos na al muscle de elopmen in he ba nacle goose. J. Exp.
Biol. 201, 827–836. (doi:10.1242/jeb.201.6.827)
65. Guglielmo CG. 2018 Obese supe a hle es: a - ueled mig a ion in bi ds and ba s. J. Exp. Biol. 221, jeb165753. (doi:10.1242/jeb.165753)
66. Peig J, G een AJ. 2009 New pe spec i es o es ima ing body condi ion om mass/leng h da a: he scaled mass index as an al e na i e me hod.
Oikos 118, 1883–1891. (doi:10.1111/j.1600-0706.2009.17643.x)
67. Gi aldo‐Deck LM, Goymann W, Sa a i I, Dawson DA, S ocks M, Bu ke T, Lank DB, Küppe C. 2020 De elopmen o in aspeci ic size a ia ion in
black coucals, whi e-b owed coucals and u s om ha ching o ledging. J. A ian Biol. 51. (doi:10.1111/ja .02440)
68. S ie A, Romes aing C, Schull Q, Le ol E, Robin JP, Roussel D, Bize P. 2017 How o measu e mi ochond ial unc ion in bi ds using ed blood cells:
a case s udy in he king penguin and pe spec i es in ecology and e olu ion. Me hods Ecol. E ol. 8, 1172–1182. (doi:10.1111/2041-210X.12724)
69. S ie A e al. 2013 A ian e y h ocy es ha e unc ional mi ochond ia, opening no el pe spec i es o bi ds as animal models in he s udy o
ageing. F on . Zool. 10, 33. (doi:10.1186/1742-9994-10-33)
70. S ie A, Reiche S, C iscuolo F, Bize P. 2015 Red blood cells open p omising a enues o longi udinal s udies o ageing in labo a o y, non-model
and wild animals. Exp. Ge on ol. 71, 118–134. (doi:10.1016/j.exge .2015.09.001)
71. Ruuskanen S, Hsu BY, Heinonen A, Vainio M, Da as VM, Sa aude T, Rokka A. 2018 A new me hod o measu ing hy oid ho mones using nano-
LC-MS/MS. J. Ch oma og . B 1093–1094, 24–30. (doi:10.1016/j.jch omb.2018.06.052)
72. MacLean B e al. 2010 Skyline: an open sou ce documen edi o o c ea ing and analyzing a ge ed p o eomics expe imen s. Bioin o ma. Ox .
Engl. 26, 966–968. (doi:10.1093/bioin o ma ics/b q054)
73. R Co e Team. 2022 R: A language and en i onmen o s a is ical compu ing. Vienna, Aus ia: R Founda ion o S a is ical Compu ing. See h ps://
www.R-p ojec .o g/.
74. Good ich B, Gab y J, Ali I, B illeman S. 2022 Bayesian applied eg ession modeling ia S an. R package e sion 2.21.3. See h ps://mc-s an.o g/
s ana m.
75. Mu h C, O a ecz Z, Gab y J. 2018 Use - iendly Bayesian eg ession modeling: a u o ial wi h s ana m and shinys an. Quan . Me hods Psychol.
14, 99–119. (doi:10.20982/ qmp.14.2.p099)
76. Llewelyn H. 2019 Replacing p- alues wi h equen is pos e io p obabili ies o eplica ion—when possible pa ame e alues mus ha e
uni o m ma ginal p io p obabili ies. PLoS One 14, e0212302. (doi:10.1371/jou nal.pone.0212302)
77. Wickham H. 2016 Ggplo 2: elegan g aphics o da a analysis. Cham, Swi ze land: Sp inge . (doi:10.1007/978-3-319-24277-4)
78. Ruuskanen S e al. 2019 T ansgene a ional endoc ine dis up ion: does elemen al pollu ion a ec egg o nes ling hy oid ho mone le els in a
wild songbi d? En i on. Pollu . 247, 725–735. (doi:10.1016/j.en pol.2019.01.088)
79. Mu ei A, Kuzela S, Nelson BD. 1989 Con ol o mi ochond ial ansc ip ion by hy oid ho mone. Eu . J. Biochem. 180, 235–240. (doi:10.1111/j.
1432-1033.1989. b14638.x)
80. Fishe MO, Nage RG, Monaghan P. 2006 Compensa o y g ow h impai s adul cogni i e pe o mance. PLoS Biol. 4, e251. (doi:10.1371/jou nal.
pbio.0040251)
81. Ha CE, Bhaga an NV. 2023 Chap e 11 - Ca bohyd a e me abolism I: glycolysis and he ica boxylic acid cycle. In Essen ials o medical
biochemis y (eds CE Ha, NV Bhaga an), pp. 203–227, 3 d edn. San Diego, CA: Academic P ess. (doi:10.1016/B978-0-323-88541-6.00030-2)
82. Nol i-Donegan D, B aganza A, Shi a S. 2020 Mi ochond ial elec on anspo chain: oxida i e phospho yla ion, oxidan p oduc ion, and
me hods o measu emen . Redox Biol. 37, 101674. (doi:10.1016/j. edox.2020.101674)
83. Eya JC, Ashame MF, Pome oy CF, Manning BB, Pe e son BC. 2012 Gene ic a ia ion in eed consump ion, g ow h, nu ien u iliza ion e iciency
and mi ochond ial unc ion wi hin a a med popula ion o channel ca ish (Ic alu us punc a us). Comp. Biochem. Physiol. B Biochem. Mol. Biol.
163, 211–220. (doi:10.1016/j.cbpb.2012.05.019)
84. Eya JC, Ashame MF, Pome oy CF. 2011 Associa ion o mi ochond ial unc ion wi h eed e iciency in ainbow ou : die s and amily e ec s.
Aquacul u e 321, 71–84. (doi:10.1016/j.aquacul u e.2011.08.037)
85. Be mejo-Nogales A, Calduch-Gine JA, Pé ez-Sánchez J. 2015 Un a eling he molecula signa u es o oxida i e phospho yla ion o cope wi h
he nu i ionally changing me abolic capabili ies o li e and muscle issues in a med ish. PLoS One 10, e0122889. (doi:10.1371/jou nal.pone.
0122889)
86. Lucassen M, Schmid A, Ecke le LG, Pö ne HO. 2003 Mi ochond ial p oli e a ion in he pe manen s. empo a y cold: enzyme ac i i ies and
mRNA le els in An a c ic and empe a e zoa cid ish. Am. J. Physiol. Regul. In eg . Comp. Physiol. 285, R1410–20. (doi:10.1152/ajp egu.00111.
2003)
17
oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417
Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024
87. O czewska JI, Ha leben G, O’B ien KM. 2010 The molecula basis o ae obic me abolic emodeling di e s be ween oxida i e muscle and li e o
h eespine s icklebacks in esponse o cold acclima ion. Am. J. Physiol. Regul. In eg . Comp. Physiol. 299, R352–64. (doi:10.1152/ajp egu.00189.
2010)
88. O’Dowd C, Mo he sill CE, Cai ns MT, Aus in B, Lyng FM, McClean B, Talbo A, Mu phy JEJ. 2009 Gene exp ession and enzyme ac i i y o
mi ochond ial p o eins in i adia ed ainbow ou (Onco hynchus mykiss, Walbaum) issues in i o. Radia . Res. 171, 464–473. (doi:10.1667/
RR1484.1)
89. Soa es SS, Gu ié ez-Me ino C, Au eliano M. 2007 Mi ochond ia as a a ge o deca anada e oxici y in Spa us au a a hea . Aqua . Toxicol. 83,
1–9. (doi:10.1016/j.aqua ox.2007.03.005)
90. S ie A, Bize P, Hsu BY, Ruuskanen S. 2019 Plas ic bu epea able: apid adjus men s o mi ochond ial unc ion and densi y du ing ep oduc ion
in a wild bi d species. Biol. Le . 15, 20190536. (doi:10.1098/ sbl.2019.0536)
91. Me cal e NB, Monaghan P. 2001 Compensa ion o a bad s a : g ow now, pay la e . T ends Ecol. E ol. 16, 254–260. (doi:10.1016/s0169-
5347(01)02124-3)
92. Pee e s RP, Visse TJ. 2000 Me abolism o hy oid ho mone. In Endo ex (eds KR Feingold e al.). Sou h Da mou h, MA: MDTex .com, Inc.
93. Xu R, Huang F, Zhang S, L Y, Liu Q. 2019 Thy oid unc ion, body mass index, and me abolic isk ma ke s in eu hy oid adul s: a coho s udy.
BMC Endoc . Diso d. 19, 58. (doi:10.1186/s12902-019-0383-2)
94. Bolund E, Schielze h H, Fo s meie W. 2010 No heigh ened condi ion dependence o zeb a inch o namen s – a quan i a i e gene ic app oach.
J. E ol. Biol. 23, 586–597. (doi:10.1111/j.1420-9101.2009.01927.x)
95. Wood SN. 2020 In e ence and compu a ion wi h gene alized addi i e models and hei ex ensions. Tes 29, 307–339. (doi:10.1007/s11749-
020-00711-5)
96. Scanes CG, Radecki SV, Malamed S. 1992 Mechanisms in ol ed in he a ian pa e ns o g ow h ho mone sec e ion du ing g ow h and
de elopmen . O nis. Scand. 23, 214–221. (doi:10.2307/3676641)
97. Eales JG. 1988 The in luence o nu i ional s a e on hy oid unc ion in a ious e eb a es. Am. Zool. 28, 351–362. (doi:10.1093/icb/28.2.351)
98. Schwabl H, Lipa J. 2002 Ho monal egula ion o begging beha iou . In The e olu ion o begging: compe i ion, coope a ion and communica ion
(eds J W igh , ML Leona d), pp. 221–244. Do d ech , The Ne he lands: Sp inge . (doi:10.1007/0-306-47660-6)
99. Cossin-Se in N, S ie A, Hukkanen M, Zahn S, Viblanc VA, An ila K, Ruuskanen S. 2023 Ea ly-li e en i onmen al e ec s on mi ochond ial
ae obic me abolism: a b ood size manipula ion in wild g ea i s. J. Exp. Biol. 226, jeb245932. (doi:10.1242/jeb.245932)
100. Rome o LM, Wing ield JC. 2015 Tempes s, poxes, p eda o s, and people: s ess in wild animals and how hey cope. Ox o d, UK: Ox o d Uni e si y
P ess. (doi:10.1093/acp o :oso/9780195366693.001.0001)
101. Robson H, Sieble T, Shale SM, Williams GR. 2002 In e ac ions be ween GH, IGF-I, glucoco icoids, and hy oid ho mones du ing skele al
g ow h. Pedia . Res. 52, 137–147. (doi:10.1203/00006450-200208000-00003)
102. Roo AW, Shulman D, Roo J, Diamond F. 1986 The in e ela ionships o hy oid and g ow h ho mones: e ec o g ow h ho mone eleasing
ho mone in hypo- and hype hy oid male a s. Ac a Endoc inol. Suppl. (Copenh.) 279, 367–375. (doi:10.1530/ac a.0.112S367)
103. Oe ele M, Hau M, Ruuskanen S, Casag ande S. 2024 Da a om: Mi ochond ial unc ion is enhanced by hy oid ho mones du ing zeb a inch
de elopmen [Da ase ]. D yad. (doi:10.5061/d yad.zpc866 d1)
104. Oe ele M, Hau M, Ruuskanen S, Casag ande S. 2024 Supplemen a y ma e ial om: Mi ochond ial unc ion is enhanced by hy oid ho mones
du ing zeb a inch de elopmen . Figsha e. (doi:10.6084/m9. igsha e.c.7349588)
18
oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417
Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024