Lack o e idence o a consis en
di e en ial impac o ail and
unnel handling on ma ke s o
wel a e in labo a o y mice
Neele Meye , Rebekka Go schalk, Lena Jakobi, Anna-Ma ia Schönho & Chadi Touma
Di e en handling me hods o labo a o y mice ha e been in ensely deba ed in ligh o e ining animal
husband y. Se e al s udies claim ha ail handling is a e si e, while unnel handling seems o ha e a
lesse impac on animal wel a e. Howe e , mos o hese s udies in es iga ed he e ec o handling
pe o med in an unusually high equency and p olonged du a ion, no ma ching labo a o y ou ines.
The e o e, he aim o ou s udy was o in es iga e he impac o weekly cage change using ail e sus
unnel handling on male and emale C57BL/6J and CD-1 mice. Locomo ion and explo a o y ac i i y as
well as anxie y- ela ed beha iou we e measu ed. Mo eo e , he animals’ in e es in social pa ne s
and social no el y as well as olun a y in e ac ion wi h he handle we e assessed. Reac i i y and
epea ed ac i a ion o he HPA axis we e moni o ed using co icos e one le els and ad enal gland
and hymus weigh s. Only e y ew o he measu ed beha iou al and s ess physiological pa ame e s
di e ed signi ican ly be ween he wo handling g oups, wi h a ying di ec ion. Ou comp ehensi e
analysis could hus e eal no consis en e idence suppo ing he supe io i y o one me hod o e he
o he in e ms o wel a e o he handled mice.
Handling me hods o labo a o y mice a e an impo an and in ensely deba ed opic in ligh o he e inemen
o animal husband y and es ing. Re inemen , one o he 3Rs, ocuses on imp o ing condi ions o animals in
esea ch by ad ancing expe imen al p ocedu es and imp o ing he housing en i onmen 1,2. Hence, limi ing
exposu e o s ess ul e en s du ing ou ine handling is a c i ical aspec impac ing animal wel a e. Especially in
labo a o y mice, he mos widely used e eb a e species in biological and biomedical esea ch3, implemen ing
e inemen is c ucial. The e o e, di e en handling me hods o ansloca ing mice o a no el cage o an
expe imen al se up ha e been in es iga ed in ecen yea s. The mos common handling me hod, i.e., handling
mice by hei ails, has become less popula due o conce ns abou a possible impac on animal wel a e4. As a
esul , p esumably less a e si e me hods, such as unnel handling, ha e been ad oca ed. Findings suppo ing
he unnel handling me hod ha e been published, showing ha unnel-handled mice showed mo e olun a y
in e ac ion wi h he handling de ice han ail-handled mice4–13. Addi ionally, an inc ease in explo a o y
beha iou and a dec ease in anxie y- ela ed beha iou we e demons a ed a leas in some s udies4,7–10,12,14.
In e es ingly, hese e ec s also seemed o pe sis when he animals we e subjec ed o mo e in asi e expe imen al
p ocedu es, such as injec ions o o al ga age4,7,8,10.
Howe e , a no ewo hy d awback o many o hese s udies is he applied equency and du a ion o handling.
Mos ly, animals we e handled in high equencies (daily o nine subsequen days) and o long du a ions (e.g.,
30s) ha do no e lec ou ine labo a o y p ocedu es. Fewe s udies ha e applied handling equencies and
du a ions compa able o he e e yday lab and animal ca e wo k o compa ed low and highe equencies/
du a ions, yielding inconsis en esul s7,11,13,15–18. Fo example, animals ou inely handled by ail showed mo e
agi a ion and we e less easy o handle (by cup handling) ollowing anspo a ion compa ed o unnel-handled
mice17. In compa ison o ou ine handling by ail using o ceps, unnel-handled C57BL6/J mice p oduced mo e
pups and had ewe p e-weaning li e losses15, while no di e ences in b eeding p oduc i i y we e ound in
ail- and unnel-handled BALB/cJ and CD-1-IGS mice16. Rega dless o handling du a ion, emales handled by
ail in e ac ed less wi h he expe imen e in he olun a y in e ac ion es han unnel-handled mice7. Rega ding
beha iou al es ou comes, one s udy ound no handling- ela ed al e a ions in anxie y- ela ed beha iou in he
Ele a ed Plus-Maze es (EPM) when handled on a ou ine basis (C57Bl/6J & BALB/cJ)18, while ano he ound
ail-handled emales (C57Bl/6JOla/Hsd) spending less ime on he open a ms compa ed o unnel-handled
Depa men o Beha iou al Biology, Osnab ück Uni e si y, Osnab ück, Ge many. email:
[email p o ec ed]; [email p o ec ed]
OPEN
Scien i ic Repo s | (2025) 15:21534 1
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s
indi iduals7. In addi ion, ou ine handling in some cases led o s ain-dependen di e ences, e.g., ega ding
locomo ion in he Open Field es 18 and anxie y- ela ed beha iou in he EPM13 o sex and/o handling-
equency dependen changes in wel a e- ela ed pa ame e s11.
Al hough he men ioned published s udies mos ly claim ha ail handling is mo e s ess ul han unnel
handling, he inclusion o physiological ma ke s o s ess and anxie y induced by he di e en handling me hods
is la gely missing. The e o e, e alua ing ho monal s ess esponses owa ds ei he handling me hod would gi e
impo an addi ional insigh s in o possible impac s on animal wel a e. The spa se e idence ha is cu en ly
a ailable shows se e al disc epancies11,13,14,19 and is hus no conclusi e.
Hence, in ou s udy, in addi ion o a numbe o ele an beha iou al es s in es iga ing locomo ion,
explo a ion, anxie y- ela ed and social beha iou , an in-dep h analysis o physiological ma ke s o acu e and
ch onic s ess esponses was pe o med in o de o in es iga e he impac o ou ine handling me hods ( ail s.
unnel handling) on animal wel a e o labo a o y mice o bo h sexes. We selec ed wo commonly used s ains
o ou s udy, one inb ed (C57BL/6J; B6) and ou b ed (CD-1), as i is known ha di e en gene ic backg ounds
migh show di e ences in beha iou al and physiological eac ions20–24. We hypo hesised di e en ial handling-
ela ed esponses in explo a ion, anxie y- ela ed and social beha iou s as well as in s ess esponses in bo h
s ains and sexes. The expe imen al imeline and he used beha iou al and s ess physiological pa adigms a e
ou lined in Fig.1.
Resul s
The esul s p esen ed in his sec ion ocus on handling- ela ed e ec s and he in e ac ion o handling wi h o he
ac o s. Apa om hese e ec s, some sex di e ences we e de ec ed. As hose a e no he main ocus o he s udy,
hese a e p esen ed in he co esponding Supplemen a y Ma e ial (Supplemen a y Tables S1–S8).
Handling ime was measu ed du ing cage changes ( ime om opening he cage un il he las mouse en e ed
he new cage wi h all ou paws). O e all, unnel handling ook longe han ail handling (abou 40s. s. 10s.
o he wo mice pe cage). Handling imes o he animals did no change much o e he en-week s udy pe iod.
De ails a e p esen ed in Supplemen a y Table S9 and he Me hods sec ion.
Beha iou al es s
Open ield es
The Open Field (OF) es was pe o med o assess locomo o ac i i y and explo a ion. O e all, only a
ew s a is ically signi ican di e ences we e obse ed be ween ail- and unnel-handled mice (Fig. 2 and
Supplemen a y Table S1). In C57BL/6J (B6) mice, unnel-handled mice a elled a g ea e dis ance compa ed
o ail-handled mice (main e ec o handling: F1,49 = 17.735, p < 0.001). In addi ion, ail-handled B6 mice spen
mo e ime in he cen e o he OF compa ed o hei unnel-handled coun e pa s (main e ec o handling:
F1,49 = 5.114, p = 0.028). In CD-1 mice, no handling- ela ed di e ences we e ound in any o he analysed
pa ame e s o he OF es (Fig.2 and Supplemen a y Table S1).
Ligh –da k box es
The Ligh –Da k Box (LD) es was used o in es iga e anxie y- ela ed beha iou o mice handled by ail o
unnel. In B6 mice, no signi ican e ec o handling me hod was obse ed in any o he analysed pa ame e s
(Fig.2 and Supplemen a y Table S2). In CD-1 mice, ail-handled animals spen signi ican ly mo e ime in he
ligh compa men han unnel-handled indi iduals (main e ec o handling: F1,48 = 4.629, p = 0.036; Fig.2 and
Supplemen a y Table S2).
Sociabili y and social no el y es
The Sociabili y and Social No el y (S-SN) es was pe o med o in es iga e he in e es o he mice in no el social
s imuli. Du ing he Sociabili y phase, i was es ed whe he he animals p e e ed an un amilia social pa ne
(un amilia male o he same s ain p esen ed unde a wi e mesh cup) o e an un amilia objec (wi e mesh cup
wi hou a social s imulus animal). Rega ding handling- ela ed e ec s, in B6 mice no di e ences be ween ail-
and unnel-handled mice we e de ec ed in he assessed pa ame e s (Supplemen a y Table S3) and in CD-1 mice,
only he dis ance a elled was a ec ed (F1,48 = 8.512, p = 0.005), e ealing a g ea e dis ance a elled in unnel-
Fig. 1. Expe imen al imeline. S a ing a 6weeks o age, animals we e ou inely handled once weekly o
10weeks, ei he by he ail o unnel handling me hod. A e wa ds, beha iou al and s ess physiological
assessmen s we e pe o med o 3weeks. Body weigh was measu ed a 10, 15, 17 and 18weeks o age. AHN,
App oach o handle and no el y es , BW, body weigh , FCM, aecal co icos e one me aboli e analysis,
LD, Ligh –Da k Box es , H, handling, OF, Open Field es , PNW, pos -na al week, SRT, s ess eac i i y es
assessing HPA axis eac i i y, S-SN, Sociabili y and Social No el y es .
Scien i ic Repo s | (2025) 15:21534 2
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/
compa ed o ail-handled mice. Tes s a is ics o all o he analysed pa ame e s a e p esen ed in Supplemen a y
Table S3.
In he Social No el y phase i was es ed whe he he mice p e e ed a no el social s imulus (un amilia mouse
o he same s ain placed unde he p e iously emp y wi e mesh cup) o e a amilia one. Mos pa ame e s
analysed did no show a handling ela ed di e ence (Supplemen a y Table S4). Howe e , ail-handled B6 mice
showed a signi ican ly highe p e e ence o he no el compa ed o he amilia social pa ne (main e ec o
handling: F1,47 = 7.073, p = 0.011; Fig.2) in compa ison o unnel-handled B6 mice. Such a di e ence was no
seen in CD-1 mice (F1,45 = 1.499, p = 0.227; Fig.2 and Supplemen a y Table S4).
App oach o handle and no el y (AHN) es
The AHN es was pe o med wice a e 10weeks o handling (see expe imen al imelines in Figs.1 and 3) in
he home cage o he animals. The di ec in e ac ion o he mouse wi h he glo ed hand o he expe imen e was
in e p e ed o e lec in e es o a oidance o he expe imen e , while in e ac ion wi h an un amilia scen ( ood-
ela ed baking a omas), e lec ed in e es o a oidance o a no el handling-un ela ed s imulus. In his es , di ec
in es iga ion was de ined as he nose o he mouse being wi hin one head-leng h o he expe imen e ’s hand o
he co on swab p esen ing he scen . The animals we e allowed o in es iga e he hand o he expe imen e and
subsequen ly an unknown scen ( ial 1&2). A e wa ds, bo h animals o he cage we e handled o 30s each
acco ding o hei assigned handling me hod, ollowed by ano he p esen a ion o he expe imen e s’ hand and a
new, un amilia scen ( ial 3 & 4). We expec ed ha animals would a oid he hand, i i was pe cei ed as a e si e
and ha his e ec would be s onge di ec ly a e a handling e en . As he pai -housed animals we e es ed
simul aneous in hei home cage, he da a was a e aged by cage.
A linea mixed model analysis was applied, wi h handling me hod, sex, ial (be o e/a e handling) and
s imulus (hand/scen ) as ixed ac o s and cage and session as andom ac o s.
Fig. 3. Schema ic o e iew o he App oach o Handle and No el y (AHN) es . The es was pe o med in he
home cage o he mice ( wo animals housed pe cage). Be o e s a ing he es , he cage lid and nes ing ma e ial
we e emo ed (acclima isa ion). The AHN es consis ed o ou ials, wo be o e and wo a e handling he
mice wi h he assigned me hod. The AHN es was pe o med wice wi h a b eak o h ee days be ween he wo
es sessions.
Fig. 2. Resul s o he beha iou al es s o C57BL/6J (B6; op panels), and CD-1 mice (bo om panels) ei he
handled by ail o unnel. Key esul s o he Open Field es (OF; dis ance a elled, ime in he cen e), he
Ligh –Da k Box es (LD; ime in and en ies o he ligh compa men ) and he Social No el y phase o he
Sociabili y and Social No el y es (S-SN; p e e ence o no el social pa ne ) a e p esen ed. Da a a e depic ed
as means + SD and indi idual alues o each mouse (males: ci cles, emales: iangles). S a is ics: wo-way
ANOVA wi h handling me hod and sex as ixed ac o s. Handling- ela ed di e ences a e p esen ed in he
g aphs: *p < 0.05, ***p < 0.001; n = 26–27 pe g oup.
Scien i ic Repo s | (2025) 15:21534 3
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/
In B6 mice, a handling me hod x ial in e ac ion (F1,189 = 4.618, p = 0.033) was ound (Fig.4 and Table
1). Pos hoc - es s showed ha in compa ison o ail-handled mice, unnel-handled mice spen mo e ime
in es iga ing he s imulus in all ials (be o e handling: = 3.536, p < 0.05; a e handling: = 5.687, p < 0.05
o bo h compa isons a e Bon e oni-Holm co ec ion). Mo eo e , unnel-handled mice in es iga ed he
p esen ed s imuli mo e a e being handled han be o e being handled ( = 2.891; p < 0.05 a e Bon e oni-Holm
co ec ion). Addi ionally, he e was a signi ican main e ec o handling me hod (F1,27 = 32.508, p < 0.001),
as unnel-handled B6 mice spen mo e ime in es iga ing he s imuli han ail-handled mice, while B6 mice
gene ally in es iga ed he no el scen mo e han he expe imen e ’s hand (F1,189 = 17.197, p < 0.001). Mo eo e , a
main e ec o ial (F1,189 = 5.483, p = 0.020) was de ec ed.
App oach o handle and no el y es
ANOVA Time spen in es iga ing
s imulus (hand o scen )
Fixed ac o s B6 CD-1
Handling me hod F1,27 = 32.508
p < 0.001 F1,25.489 = 0.526
p = 0.475
Sex F1,27 = 1.693
p = 0.204 F1,25.489 = 0.934
p = 0.343
T ial (be o e/a e handling) F1,189 = 5.483
p = 0.020 F1,179.789 = 23.141
p < 0.001
S imulus
(hand o scen ) F1,189 = 17.197
p < 0.001 F1,179.789 = 93.739
p < 0.001
Handling me hod x sex F1,27 = 1.876
p = 0.182 F1,25.489 = 0.471
p = 0.499
Handling me hod x ial F1,189 = 4.618
p = 0.033 F1,179.781 = 1.748
p = 0.188
Handling me hod x s imulus F1,189 = 0.632
p = 0.428 F1,179.781 = 3.048
p = 0.083
Sex x ial F1,189 = 5.055
p = 0.026 F1,179.781 = 0.217
p = 0.642
Sex x s imulus F1,189 = 1.082
p = 0.300 F1,179.781 = 0.085
p = 0.771
T ial x s imulus F1,189 = 0.274
p = 0.601 F1,179.781 = 2.765
p = 0.098
Table 1. Tes s a is ics o he linea mixed model ANOVA o he App oach o Handle and No el y (AHN)
es o C57BL6/J (B6) and CD-1 mice wi h handling me hod ( ail o unnel), sex (male/ emale), ial (be o e/
a e handling) and s imulus (expe imen e s’ hand o no el scen ) as ixed ac o s and cage (means o bo h
animals in he cage) and session as andom ac o s. S a is ically signi ican esul s a e ma ked in bold.
Fig. 4. Time spen explo ing he hand o scen du ing he App oach o Handle and No el y (AHN) es
o C57BL/6J (B6) (le panel) and CD-1 mice ( igh panel). The hand and he scen we e p esen ed o 60s
be o e and a e handling he mice using he assigned handling me hod ( ail o unnel). Da a a e depic ed as
means + SD and indi idual alues (please no e ha he e a e wo alues pe cage ep esen ing he wo es ing
sessions; males: ci cles, emales: iangles). S a is ics: linea mixed model wi h handling me hod, sex, ial
(be o e/a e ) and s imulus (hand o scen ) as ixed ac o s and cage and session as andom ac o s. Handling-
ela ed di e ences a e p esen ed in he g aphs, all s a is ical compa isons a e shown in Table 1; n = 13–14 cages
pe g oup.
Scien i ic Repo s | (2025) 15:21534 4
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/
In CD-1 mice, no signi ican e ec s o handling me hod on in es iga ing he hand o scen we e de ec ed
(Fig.4 and Table 1). The e was a main e ec o ial, showing ha he in es iga ion was longe a e being handled
han be o e being handled (F1,179.8 = 23.141, p < 0.001) and a main e ec o he p esen ed s imulus, showing ha
CD-1 mice explo ed he hand o he expe imen e longe han he scen (F1,179.8 = 93.739, p < 0.001). S a is ical
de ails can be ound in Table 1.
Physiological measu es
Body weigh
Body weigh was assessed a ou ime poin s ac oss he 10-week handling pe iod and he h ee beha iou al
es ing weeks (da a shown in Supplemen a y Table S5).
In B6 mice, a epea ed-measu es ANOVA e ealed a signi ican age x handling x sex in e ac ion (F3,147 = 3.510,
p = 0.017). Pos hoc - es s showed no handling- ela ed di e ences a he di e en measu ing ime poin s (p > 0.05
o each ime poin a e Bon e oni-Holm co ec ion), bu , as expec ed, male mice we e signi ican ly hea ie
han emale mice (p < 0.05 o each ime poin a e Bon e oni-Holm co ec ion).
In CD-1 mice, no handling ela ed di e ences in body weigh we e de ec ed (age x handling me hod:
F3,144 = 0.295,p = 0.829, Supplemen a y Table S5).
Faecal glucoco icoid me aboli es as a ma ke o s ess in esponse o handling
In B6 mice, ollowing handling, he e was a signi ican main e ec o handling me hod (F1,45 = 4.894, p = 0.032)
on aecal glucoco icoid me aboli e ( GCM) concen a ions. Tunnel-handled mice showed highe GCM le els
han ail-handled B6 mice (Fig.5 and Supplemen a y Table S6). In CD-1 mice, no s a is ically signi ican
handling- ela ed di e ences we e obse ed (F1,45 = 2.931, p = 0.094, Supplemen a y Table S6).
HPA axis eac i i y in esponse o a psychological s esso
To in es iga e HPA axis eac i i y, plasma co icos e one concen a ions we e measu ed a baseline (ini ial),
a e a 15-min es ain ( esponse) and a e a eco e y pe iod o 75min a e s esso e mina ion ( eco e y).
O e all, in bo h mouse s ains, no handling ela ed di e ences we e de ec ed by epea ed-measu es ANOVA
(Fig.6 and Supplemen a y Table S7).
O gan weigh as a ch onic s ess ma ke
In B6 and CD-1 mice, he weigh o he ad enal glands (mean o le and igh ad enal) and he hymus, bo h
ela i e o body weigh , we e no signi ican ly a ec ed by handling me hod (Fig.7), al hough he expec ed sex
di e ences we e ound (hea ie in emales, Supplemen a y Table S8).
Discussion
As handling is one o he labo a o y ou ine p ocedu es ha all labo a o y mice expe ience egula ly, i is
impo an o minimise po en ial nega i e impac s on animal wel a e. The e o e, ou s udy aimed o in es iga e
whe he handling mice by ail o unnel, a a equency and du a ion usually applied du ing ou ine housing and
es ing in biomedical esea ch, a ec s he animals di e en ly ega ding beha iou al and physiological ma ke s
o animal wel a e.
O e all, o bo h mouse s ains we ound e y ew s a is ically signi ican and a he inconsis en handling-
ela ed e ec s on he measu ed beha iou al and physiological pa ame e s. Explo a o y beha iou , locomo ion
and anxie y- ela ed beha iou showed li le o no handling- ela ed al e a ions. In ou OF es ( i e minu es es
du a ion), B6 unnel-handled mice a elled a g ea e dis ance han ail-handled indi iduals, while ail-handled
indi iduals spen mo e ime in he cen e o he a ena. A p e ious s udy18 also applying less equen handling
(b ie handling wice a week o cage changes) ound no signi ican di e ence in locomo o ac i i y in he OF
Fig. 5. Faecal glucoco icoid me aboli e ( GCM) concen a ions (ng/0.05g aeces) as a physiological s ess
ma ke in esponse o handling by ail o unnel in C57BL/6J (B6; le panel) and CD-1 ( igh panel) mice.
Da a a e p esen ed as means + SD and indi idual alues (males: ci cles, emales: iangles. S a is ics: wo-way
ANOVA wi h handling me hod and sex as ixed ac o s. Handling- ela ed di e ences a e p esen ed in he
g aphs: *p < 0.05; n = 23–26 pe g oup.
Scien i ic Repo s | (2025) 15:21534 5
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/

es (60min es du a ion) be ween emale ail- and unnel-handled B6 mice, bu obse ed signi ican e ec s
o BALB/c mice. Simila ly, he locomo o ac i i y was sligh ly inc eased in unnel-handle CD-1 mice in he
sociabili y phase o ou S-SN es . I seems possible ha le ing he animals olun a ily en e he a ena (eme ging
om he unnel) e sus placing hem down by hei ail impac s he ini ial locomo o ac i i y in he beha iou al
es s, which could a leas pa ially accoun o he obse ed di e ences. S udies applying highe handling
equencies o du a ions also ound simila e ec s ega ding locomo ion in he OF, ye con as ing esul s o
he ime spen in he cen e o he a ena7,8 ha we e s ill obse ed 3weeks a e he o se o daily handling9,12.
While his migh be explained by handling equency, o he ac o s such as illumina ion in ensi y o he a ena,
loca ion o placemen o he mouse, sex and s ain a e impo an ac o s as well and canno be excluded.
Anxie y- ela ed beha iou was measu ed in he Ligh –Da k Box (LD) es . We ound no signi ican di e ences
be ween he handling g oups in any ele an pa ame e in B6 mice. These indings a e in line wi h ano he ecen
s udy ha handled mice wice a week, whe e B6 mice also did no show handling- ela ed e ec s in he Ele a ed-
Plus Maze (EPM) es 13. Ye , he ail-handled CD-1 mice in ou s udy spen mo e ime in he ligh compa men
compa ed o hei unnel-handled coun e pa s, indica ing less anxie y- ela ed beha iou . Howe e , he o he
pa ame e s es ed in he LD es we e no signi ican ly al e ed by handling me hod. Con as ing o ou esul s,
in a s udy by Gou eia and Hu s , anxie y- ela ed beha iou was shown o be inc eased in ail-handled mice
compa ed o unnel-handled indi iduals, independen o handling equency7. This migh be explained by
a di e en s ain being used (BALB/cOlaHsd), mice being es ed du ing he da k phase and p obably mos
impo an ly, by placemen in o he EPM acing he open a m s acing he closed a m7. S udies in es iga ing a
Fig. 6. Plasma co icos e one concen a ions (ng/ml) o C57BL/6J (B6; op panel) and CD-1 mice (bo om
panel). Co icos e one le els a e shown a baseline (ini ial), a e a 15-min es ain ( esponse) and a e
a eco e y pe iod o 75min a e s esso e mina ion ( eco e y). Da a a e depic ed as means + SD and
indi idual alues (males: ci cles, emales: iangles). S a is ics: epea ed-measu es ANOVA wi h handling
me hod and sex as be ween-subjec ac o s. No signi ican handling- ela ed di e ences we e de ec ed;
n = 20–27 pe g oup.
Scien i ic Repo s | (2025) 15:21534 6
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/
longe du a ion and highe equency o handling on anxie y- ela ed beha iou , mainly ound ha ail-handled
indi iduals showed inc eased anxie y- ela ed beha iou in he LD14 and EPM es (e.g.,4,7,12) o p oduced
inconclusi e esul s8.
In addi ion o explo a ion and anxie y- ela ed beha iou s, social beha iou was assessed in he Sociabili y
and Social No el y es . While gene al in e es in an un amilia social pa ne (un amilia male mouse o he
same s ain) measu ed in he Sociabili y Phase was no signi ican ly a ec ed by handling me hod, ail-handled
B6 mice showed a g ea e p e e ence o a no el conspeci ic in he Social No el y Phase han hei unnel-handled
coun e pa s, indica ing a s onge in e es in social no el y, which is expec ed o a socially li ing species25.
O e all, social beha iou , as measu ed in his es , was no s ongly al e ed by handling me hod.
In he App oach o Handle and No el y (AHN) es , unnel-handled B6 mice spen mo e ime in es iga ing
he p esen ed s imuli (hand o scen ) han ail-handled mice and hey also inc eased hei explo a ion a e being
handled. This is in line wi h esul s ound in o he s udies (e.g.,4–13). I seems ha ail-handled B6 mice migh
pe cei e he hand as mo e o a h ea compa ed o unnel handled mice. In con as , CD-1 mice we e a he
una ec ed by handling me hod in he AHN es . Animals o bo h handling g oups and s ains in es iga ed
he s imulus mo e a e being handled han be o e, indica ing a possible ac i a ing e ec o being handled.
The in oduc ion o a handling-un ela ed s imulus (an un amilia baking a oma on a co on swab) o he es
in ou s udy was a new app oach o disen angling he possible a oidance o he handle and he a oidance o
gene al no el y in oduced o he cage. In e es ingly, independen o handling me hod, B6 mice spen mo e ime
in es iga ing he scen compa ed o he hand, while his was he e e se in CD-1 mice. I migh he e o e be
specula ed, ha B6 mice in gene al a e mo e anxious owa ds handling- ela ed han handling-un ela ed s imuli.
Fo in e p e ing he esul s, i has o be kep in mind ha in s udies by o he esea che s usually he handling
Fig. 7. O gan weigh ela i e o body weigh (bw) o ad enal glands (mean o he le and igh ad enal gland)
and hymus o C57BL/6J (B6; op panels) and CD-1 mice (bo om panels). Da a a e depic ed as means + SD
and indi idual alues o each mouse (males: ci cles, emales: iangles). S a is ics: wo-way ANOVA wi h
handling me hod and sex as ixed ac o s. No signi ican handling- ela ed di e ences we e ound; n = 21–22 pe
g oup.
Scien i ic Repo s | (2025) 15:21534 7
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/
de ice (i.e., he hand o he unnel) is p esen ed, and he ime in es iga ing ei he hand o unnel is e alua ed
(e.g.,4). We changed his p o ocol as we hink ha he unnel, in gene al, is likely o be mo e a ac i e o he
animals, as i p o ides shel e and mo e space o explo a ion han he expe imen e ’s hand. This a gumen
is suppo ed by indings om a s udy p esen ing he unnel and he hand, i espec i e o handling me hod,
whe e he unnel was explo ed mo e han he hand13. Thus, he p e iously published di e ences be ween ail-
and unnel-handled animals migh ha e simply been due o a gene al di e ence in in e es be ween hand and
unnel. Ye , simila o ou app oach, a s udy by Hende son and colleagues10 also only in es iga ed he in e ac ion
wi h he hand. He e, animals handled by unnel spen mo e ime explo ing he hand compa ed o ail-handled
indi iduals on days i e and nine o daily handling bu no on day one10. Howe e , he mouse s ain in es iga ed
in ha s udy was di e en (BALB/c) and handling du a ion and equency we e highe 10. Ano he s udy ha
handled CD-1 mice on a ou ine basis using cup handling in es iga ed he e ec s o a ail- o non- ail es ain
echnique in a simila design also only p esen ing he expe imen e ’s hand be o e and a e es ain and ound
no es ain - ela ed di e ence in olun a y in e ac ion wi h he handle 17. A di e ence ha has o be kep in
mind is he iming o ou AHN and he olun a y in e ac ion es (VIT) in p e ious s udies (see ci a ions abo e).
In ou s udy, animals we e accus omed o handling o se e al weeks, while in he ci ed s udies, he VIT was
mos ly pe o med se e al imes, s a ing wi h he onse o applying he di e en handling me hods. The e o e,
in ligh o he di e en app oaches, di ec compa ison and in e p e a ion o he esul s is di icul . O e all i
seems ha in ou s udy, ail-handled B6 mice show a mo e cau ious esponse owa ds he handle , while CD-1
mice do no show his di e en ial esponse. Howe e , in gene al, o e -in e p e a ion o hese indings should be
a oided. Unlike he classical beha iou al es s (OF, LD, EPM, S-SN e c.), he alidi y o he VIT and AHN has no
been p o en ye , o ins ance by showing a escue o he educed ime explo ing he expe imen e ’s hand a e
anxioly ic d ug ea men in ail-handled animals.
Some aspec s ha need o be kep in mind when in e p e ing he da a p esen ed in ou s udy is ha in
con as o many s udies in es iga ing ail and unnel handling, he mice did no ha e he unnel in hei home
cage, bu i was s o ed in he ood hoppe . One s udy showed howe e , ha once B6 mice we e expe ienced in
unnel handling, he amilia i y wi h he unnel (sha ed unnel be ween cages s. home cage unnel) did no
signi ican ly a ec he in e ac ion wi h he handling de ice and did no lead o di e ences in anxie y- ela ed
beha iou in he EPM es 5. Mo eo e , in ou s udy handling ime was longe o unnel- compa ed o ail-
handled mice ( o de ails see Me hod sec ion and Table S9), which migh ha e had an impac on he beha iou al
and physiological esponses in es iga ed. Fu he mo e, all es s we e pe o med du ing he ligh -phase, which
was di e en in o he s udies.
In gene al, in ou s udy he ou comes o he classical beha iou al es s (OF, LD and S-SN) showed only a ew
e ec s o he di e en handling me hods and he di ec ion o hese e ec s we e no consis en ( unnel-handled
mice showed mo e locomo ion, while ail-handled mice showed less anxie y- ela ed beha iou and mo e in e es
in a no el social s imulus). In addi ion, he e ec s we e o en only ound in one o he wo s ains, indica ing
no gene alizable and/o s ong e ec s. In he AHN, ail-handled B6 mice showed a mo e consis en a oidance
esponse owa ds he handle , while we ound no signi ican handling- ela ed di e ences o CD-1 mice.
Rega ding he physiological pa ame e s in ol ed in he s ess esponse and wi h ele ance o assessing
animal wel a e, da a om p e ious s udies a e sca ce and do no p o ide clea esul s. Fo example, a s udy
by Ono and colleagues19 could show ha plasma co icos e one le els we e highe in ail-handled C57BL/6
compa ed o he con ol g oup, ye in BALB/c mice, unnel-handled animals had highe le els han ail-handled
indi iduals. In ou s udy, we did no ind handling- ela ed e ec s in plasma co icos e one le els (nei he a
baseline, no a e a psychological s esso ), which is also in line wi h a ecen s udy ha did no ind di e ences
in plasma co icos e one13. Howe e , ou GCM analysis e ealed ha unnel-handled B6 mice showed a sligh ly
bu signi ican ly s onge ac i a ion o he HPA axis in esponse o handling. This is di e gen o o he s udies
showing no di e ence due o handling me hod in aecal co icos e one me aboli es11,14. Howe e , in hese
s udies, HPA axis ac i a ion was no measu ed in a manne di ec ly ela ed o he handling e en 11,14.
In addi ion, he weigh o he ad enal glands and hymus, wo o gans ha a e in ol ed in he s ess esponse
and ep esen eliable ma ke s o ch onic s ess (e.g.,26), did no show any signi ican di e ences. While his is
in line wi h p e ious indings13, o he s ound an inc ease in ad enal gland weigh ollowing ail handling12. This
di e ence is likely o be explained by he highe equency and du a ion o handling (once o wice a week s.
daily handling on nine consecu i e days). Thus, om he comp ehensi e da a collec ed in ou s udy, no e idence
o a conclusi e e ec o he handling me hod on physiological s ess ma ke s was ound.
Taken oge he , ou ex ensi e and de ailed analysis did no e eal clea and consis en di e ences in
beha iou al pa e ns be ween mice handled by ail o by a unnel ha was no p esen in he home cage. Mo eo e ,
we did no obse e a s ong impac o he handling me hod on s ess physiological pa ame e s in B6 and CD-1
mice. O e all, while he e a e a ew s a is ically signi ican di e ences be ween handling g oups, hese we e only
obse ed in some o he analysed pa ame e s and no consis en pa e n a ou ing one handling me hod o e
he o he became appa en . We canno d aw di ec conclusions abou s ain di e ences, as s ains we e analysed
sepa a ely, bu he e we e sligh ly mo e handling- ela ed di e ences be ween ail- and unnel-handled B6 han
be ween ail- and unnel handled CD-1 mice. Thus, based on ou comp ehensi e e alua ion, ail- and unnel-
handling seem o ha e no consis en di e en ial impac on beha iou al and physiological ma ke s o wel a e
when applied on a ou ine basis.
Animals, ma e ials and me hods
The p esen ed wo k complies wi h cu en egula ions co e ing animal expe imen a ion in Ge many and he
Eu opean Union (Eu opean Di ec i e 2010/63/EU). The au ho s also complied wi h he ARRIVE guidelines. All
expe imen s we e announced o and app o ed by he Lowe Saxony S a e O ice o Consume P o ec ion and
Food Sa e y (LAVES, licence no. 22/00177) and he ‘Animal Wel a e O ice ’ o Osnab ück Uni e si y.
Scien i ic Repo s | (2025) 15:21534 8
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/
Animals
Male and emale mice o he C57BL/6J (B6) and CD-1 s ains we e ob ained om ou in-house b eeding
colonies. In o al, n = 112 animals we e es ed (n = 14 pe g oup; de ia ions om his sample size we e due o
echnical easons in he di e en es s. De ails can be ound in he Supplemen a y Ma e ial). Fo es ima ion
o sample size, an a p io i powe analysis was conduc ed based on esul s om p e ious s udies o ensu e he
de ec ion o biologically ele an di e ences be ween handling me hods wi h a powe o 0.8 (β = 0.20, α = 0.05
and e ec size ( ) = 0.40). Animals we e housed in open Mac olon Type II long cages (365 × 205 x 140mm (l x
w x h)) wi h bedding (Aspen wood chips, 4HK, Al omin GmbH, Lage, Ge many) and nes ing ma e ial (wood
sha ings (NBF E-011, Al omin GmbH, Lage, Ge many)). Food (hyb id pelle s (No 1328, Al omin GmbH,
Lage, Ge many)) and wa e we e p o ided ad libi um. The animal housing ooms we e kep a a 12:12h ligh –
da k cycle (ligh s on a 8 am) wi h a cons an oom empe a u e o 22 ± 2°C and ela i e humidi y o 55 ± 10%.
All es s we e pe o med in he i s h ee hou s o he ligh phase, unless s a ed o he wise.
F om weaning (a ou weeks o age) un il he s a o he expe imen (a six weeks o age), he animals we e
handled by ail-handling. S a ing a six weeks o age, mice we e kep in same-sex g oups o wo indi iduals.
Hal o he animals we e handled by ail, and he o he hal we e handled by unnel handling (de ails see below).
Wi hin each cage, he same handling me hod was applied, and he handling me hod was assigned in a andom
ashion, ye ensu ing ha all g oups consis ed o he same numbe o animals. Cages o ail- and unnel-handled
animals we e placed al e na ely in he acks. The handling unnel was s o ed in he ood hoppe o he espec i e
cage lid and was no p o ided as an en ichmen i em inside he cage, as we wan ed o ocus on e ec s o handling
independen om he unnel as cage en ichmen .
S a ing om pos na al week (PNW) six, animals we e handled once a week o 10weeks o cage change and
heal h inspec ion acco ding o hei assigned handling me hod. Al e na ing be ween weeks, mice o ei he he
ail handling o he unnel handling g oup we e handled i s . Body weigh was assessed a he age o 10, 15, 17
and 18weeks (see expe imen al imeline in Fig.1).
Handling me hods
Fou expe imen e s, equally expe ienced wi h bo h handling me hods, pe o med he handling. Fo ail
handling, mice we e gen ly picked up a he middle o hei ails, placed on he back o he glo ed expe imen e ’s
hand (glo es: Peha-so ni ile, Paul Ha mann AG, Heidenheim, Ge many), and ans e ed o a new cage o
es appa a us o beha iou al es ing. He e, hey we e li ed by he ail again and placed down gen ly.
Fo unnel handling, he unnel ( ed anslucen unnel; Ø 5cm, leng h 10cm, Plexx BV, The Ne he lands)
was placed on he bedding in he cage (while in he hand o he expe imen e ). The mice we e allowed o en e
he unnel olun a ily. I hey did no en e he unnel on hei own wi hin a couple o seconds, hey we e gen ly
guided in o he unnel by he ee hand o he expe imen e . Once he animal was inside he unnel, he open
ends we e loosely co e ed wi h bo h glo ed hands o ensu e ha he mouse s ayed in he unnel, and he mouse
was ans e ed o a new cage o he es appa a us o beha iou al es ing. He e, he unnel was il ed sligh ly
backwa ds and he mouse could olun a ily lea e he unnel.
Handling ime was also aken in o accoun and was measu ed du ing cage changes ( ime om opening he
cage un il he las mouse en e ed he new cage wi h all ou paws). O e all, unnel handling ook longe han
ail handling (abou 10s. s. 40s.), and he ime i ook o handle he animals did no change much o e he
en-week s udy pe iod (Supplemen a y Table S9). This is somewha di e en om o he s udies applying bo h
handling me hods and no epo ing his conside ably longe handling imes ( unnel handling ei he ook only
a couple o seconds longe 27 o showed a 1.7- old di e ence28). This migh be explained by he ac ha unlike in
ou s udy, he animals in hose s udies had access o he unnel in hei home cage and hus we e mo e amilia
wi h he handling de ice27. Mo eo e , he longe ime o cage change using unnel-handling in ou s udy migh
be due o he pe iod each animal was allowed o en e he unnel olun a ily. In cases, when an animal did no
en e he unnel e en when gen ly guided by he expe imen e , he expe imen e s epped back om he cage
o se e al seconds, lea ing he unnel in he cage, gi ing he animals mo e ime and hen s a ed he handling
p ocedu e again in an a emp o minimise handling-s ess, hus expanding he o al ime o he cage ans e
o hese mice.
Beha iou al measu es
Open ield es
The Open Field es (OF) was pe o med o measu e di e ences in locomo o ac i i y and explo a i e beha iou .
Each mouse was placed in he ou e pa close o he wall o he es appa a us acing away om he cen e o
he ci cula a ena (Ø 600mm, wall heigh 400mm, illumina ion abou 15lx) using hei assigned handling
me hod. The OF es las ed o i e minu es, and a e wa ds, he animals we e placed back in hei home using
hei assigned handling me hod. The OF es was eco ded and analysed using he acking so wa e ANYmaze
( e sion 7.0, S oel ing Eu ope). The pa ame e s analysed we e he o al dis ance a elled, he ime spen in he
cen e (inne 300mm o he OF a ena) and he numbe o en ies o he cen e. Bo h mice o he same cage we e
es ed simul aneously in iden ical appa a uses. The di e en handling g oups we e es ed in a coun e balanced
design. Males and emales we e es ed on consecu i e days. Fo ob ious easons, he expe imen e s we e no
blind o he handling me hod and s ain while conduc ing he es s. Howe e , he au oma ed acking so wa e
ensu ed unbiased beha iou al analyses.
Ligh –da k box es
The Ligh –Da k Box (LD) es was pe o med o assess anxie y- ela ed beha iou . The appa a us consis ed o
a da k compa men (illumina ion < 15lx; 185 × 200mm (l x w)) and a b igh ly li compa men (illumina ion
700lx; 290 × 200mm (l x w)) ha we e connec ed by a sho unnel (45 × 60mm (l x w)). The mice we e placed
Scien i ic Repo s | (2025) 15:21534 9
| h ps://doi.o g/10.1038/s41598-025-07384-w
www.na u e.com/scien i ic epo s/