JOURNAL OF BIOLOGICAL RHYTHMS / June 1998Demas, Nelson / IMMUNE FUNCTION IN DEER MICE
Pho ope iod, Ambien Tempe a u e, and
Food A ailabili y In e ac o A ec Rep oduc i e
and Immune Func ion in Adul
Male Dee Mice (Pe omyscus manicula us)
G ego y E. Demas and Randy J. Nelson1
Depa men o Psychology, Beha io al Neu oendoc inology G oup,
Depa men o Popula ion Dynamics, Di ision o Rep oduc i e Biology,
and Depa men o Neu oscience, Johns Hopkins Uni e si y, Bal imo e, MD 21218
Abs ac Win e is o en s ess ul. Inc eased ene ge ic demands in win e and
concu en educ ions in ene gy a ailabili y can lead o an ene ge ic imbalance
and comp omise su i al. To inc ease he odds o su i ing win e , indi iduals
o some non opical oden species ha e e ol ed mechanisms o enhance im-
mune unc ion in ad ance o ha sh win e condi ions. Sho day leng hs p o ide
a p oxima e cue o enhancemen o immune unc ion, an adap i e unc ional
esponse o coun e en i onmen al s ess-induced educ ion in immune unc-
ion. In he p esen s udy, pho ope iod, ambien empe a u e, and ood a ailabil-
i y we e manipula ed and ep oduc i e unc ion and cell-media ed immuni y
we e assessed in adul male dee mice (Pe omyscus manicula us). Mice main ained
in sho days eg essed hei ep oduc i e sys ems and displayed enhanced
immune unc ion compa ed o long-day animals. Reduced ood a ailabili y
ele a ed co icos e one concen a ions and supp essed ep oduc i e and im-
mune unc ion, whe eas ambien empe a u e alone had no e ec on cell-medi-
a ed immuni y. The supp essi e e ec o ood es ic ion on ep oduc i e and
immune unc ion was o e come by main aining animals in sho days. Howe e ,
sho -day, ood- es ic ed mice main ained a low ambien empe a u es dis-
played educed ep oduc i e and immune unc ion compa ed o animals main-
ained a mild empe a u es. Taken oge he , hese esul s sugges ha sho -day
enhancemen o immune unc ion can coun e ac some, bu no all, o he
immunosupp essi e e ec s o win e s esso s. These da a a e consis en wi h
he hypo hesis ha immune unc ion is enhanced in sho days o coun e ac
s ess-media ed immune supp ession occu ing du ing win e .
Key wo ds seasonali y, hy hms, s ess, immuni y, spleen, co icos e one, ad enal
INTRODUCTION
Indi iduals o mos empe a e zone oden species
s udied o da e ely mainly on pho ope iod (day
leng h) as a p ecise empo al cue o es ima e he ime
o yea . Pho ope iodic in o ma ion is used o phase
ene ge ically expensi e ac i i ies o coincide wi h ade-
qua e ene gy a ailabili y (Nelson e al., 1990). Fo
1. To whom all co espondence should be add essed.
JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 13 No. 3, June 1998 253-262
© 1998 Sage Publica ions, Inc. 253
example, b eeding is ene ge ically demanding, and
b eeding a inapp op ia e imes o he yea can com-
p omise su i al o bo h pa en s and o sp ing
(B onson, 1989). Consequen ly, indi iduals o many
species ha e e ol ed o es ic b eeding o speci ic
seasons o he yea when ene gy a ailabili y is high.
P e ious s udies o seasonal changes in mammal-
ian physiology and beha io ha e placed conside able
emphasis on seasonal luc ua ions in ep oduc ion
and ene ge ics (B onson and Heideman, 1994;
Wunde , 1992). Many s udies ha e demons a ed
s iking seasonal pa e ns o ma ing and bi h
(B onson and Heideman, 1994; B onson, 1995).
Al hough less ex ensi ely s udied, he e also exis
salien cycles o in ec ious disease and dea h (John,
1994; Lochmille e al., 1994; Nelson and Demas, 1996).
Disease p e alence is ypically inc eased du ing he
all and win e compa ed o sp ing and summe o
indi iduals o many non opical e eb a e species
(John, 1994; Lochmille e al., 1994). Many o hese
animals p esumably become sick and die om expo-
su e o ex eme wea he o s a a ion; howe e , many
animals die om oppo unis ic diseases ha seem o
o e whelm immunological de enses, p esumably a
imes when hese de enses a e comp omised.
Se e al clinical s udies ha e p o ided e idence o
educed immune unc ion and inc eased dea h a es
om in ec ious disease du ing he win e (A oke e al.,
1993; Boc o e al., 1989). Addi ionally, seasonal
changes in immune pa ame e s epo ed in ield s ud-
ies o nonhuman animals ha e ypically epo ed
educ ions in lymphoid issue mass and immune unc-
ion du ing win e compa ed o summe (John, 1994;
Lochmille e al., 1994; Nelson and Demas, 1996). Fo
example, educ ions in whi e blood cells in co on a s
(Sigmodon hispidus) (Lochmille e al., 1994) and
splenic lymphoid issue in Eu opean g ound squi els
(Ci ellus ci ellus) (Shi a che a and Hadjiolo , 1987)
occu in win e compa ed o summe . G ound squi -
els also display lowe le els o hemagglu inins aised
agains sheep ed blood cells in win e (Sidky e al.,
1972). In sho - ailed oles (Mic o us ag es is), bo h
splenic mass and splenic e icula cell coun s a e
educed in win e as compa ed o sp ing and summe
(Newson, 1962).
Pho ope iod appea s o play a c i ical ole in medi-
a ing seasonal changes in immune unc ion. Se e al
labo a o y s udies ha e epo ed pho ope iodic
changes in splenic mass in dee mice (Pe omyscus mani-
cula us) (V iend and Laube , 1973), Sy ian hams e s
(Mesoc ice us au a us) (B aina d e al., 1987; B aina d
e al., 1988; Vaughan e al., 1987), and labo a o y
s ains o a s (Ra us no egicus) (Wu man and
Weisel, 1969). In con as o ield s udies, i ually all
labo a o y s udies o pho ope iodic changes in immune
pa ame e s demons a e enhanced immune unc ion
in sho , win e -like pho ope iods. Fo example, dee
mice signi ican ly inc ease lymphocy e, neu ophil,
and whi e blood cell coun s in sho pho ope iods
(Blom e al., 1994). Inc eased lympho-p oli e a i e ac-
i i y and changes in spleen mo phology occu in ham-
s e s main ained on sho day leng hs (Champney and
McMu ay, 1991). Addi ionally, main enance in sho
days can enhance bo h cell-media ed and humo al
immuni y in dee mice (Demas and Nelson, 1996;
Demas e al., 1997; Demas and Nelson, 1998).
Recen ly, he in e ac i e e ec s o pho ope iod and
ambien empe a u e on immuni y we e examined in
dee mice (Demas and Nelson, 1996). B ie ly, animals
we e main ained in long o sho days and in mild o
low ambien empe a u es. To al se um immuno-
globulin (Ig) G concen a ions we e ele a ed in sho -
day mice main ained a mild empe a u es compa ed
o long-day mice. Howe e , long-day mice kep in low
ambien empe a u es had educed IgG, whe eas
sho -day mice main ained in low empe a u es had
IgG concen a ions compa able o long-day animals
main ained in mild ambien empe a u es. Taken o-
ge he , hese esul s appea o esol e he disc epancy
be ween ield s udies demons a ing educed immune
unc ion in win e compa ed o summe and labo a-
o y s udies demons a ing enhanced immune unc-
ion in sho , win e -like pho ope iods compa ed o
long, summe -like day leng hs. The ne e ec o ele-
a ed immune unc ion in sho days appea s o be o
coun e ac he supp essi e e ec s o en i onmen al
s esso s, such as low ambien empe a u es, on
immune unc ion.
Al hough pho ope iod is he p ima y cue o sea-
sonally b eeding animal species, many o he en i on-
men al ac o s, including empe a u e, humidi y, ain-
all, and ood a ailabili y, a y on a seasonal basis, and
some o hese ac o s may be pe cei ed as s ess ul
(B onson, 1989; Sapolsky, 1992). The p esen expe i-
men was designed o examine ano he po en ial sea-
sonal s esso , educed ood a ailabili y, and i s in e -
ac ion wi h pho ope iod and ambien empe a u e o
a ec ep oduc i e and immune unc ion in dee mice.
In common wi h exposu e o low ambien empe a-
u es, educed ood a ailabili y e okes inc eased glu-
coco icoid sec e ion in mammals (Mu phy and Wide-
man, 1992). P e ious esea ch (Demas and Nelson,
254 JOURNAL OF BIOLOGICAL RHYTHMS / June 1998
1996) examined o al an ibody concen a ions; how-
e e , his measu e does no necessa ily e lec immune
unc ion pe se because he immune sys em emains
unchallenged. To assess immune unc ion di ec ly,
splenocy e p oli e a ion o he T-cell mi ogen, conca-
na alin A (Con A), was measu ed in he p esen s udy.
I was p edic ed ha ood es ic ion, in common wi h
educed ambien empe a u es, ac s as an en i on-
men al s esso leading o inc eased glucoco icoid
sec e ion and immune supp ession.
MATERIALS AND METHOD
Animals and Housing Condi ions
Eigh y adul (> 60 days o age) male dee mice
(Pe omyscus manicula us bai dii) we e ob ained om
ou labo a o y b eeding colony. This colony is de i ed
om animals om he Pe omyscus Gene ic S ock Cen-
e a he Uni e si y o Sou h Ca olina. These animals
a e descendan s o animals o iginally apped nea
Eas Lansing, Michigan (la i ude = 42º 51′ N).
Dee mice we e weaned a 21 days o age and
housed wi h same-sex siblings. Two weeks p io o he
ini ia ion o he expe imen , all animals we e indi-
idually housed in polyp opylene cages in colony
ooms unde a cycle o 16 h ligh and 8 h da k (L16:D8;
ligh s on a 0600 h EST). Rela i e humidi y was main-
ained a 50 ± 5%. Food (Agway P olab 1000, Sy acuse,
NY) and ap wa e we e p o ided ad libi um h ough-
ou he expe imen unless o he wise no ed. P io o
he s a o he expe imen , animals (> 60 days) we e
weighed o he nea es 0.1 g daily o 2 weeks o
es ablish baseline body mass. Food consump ion was
also assessed daily by weighing he ood blocks
emaining in he hoppe , and an a e age daily ood
in ake was de e mined o each animal.
Expe imen al Condi ions
All o he expe imen al animals we e andomly
assigned o one o ou expe imen al g oups: 1)
LD/20ºC animals (n = 20) we e housed in a long-day
pho ope iod (L16:D8) wi h colony oom empe a u e
kep cons an a 20 ± 1ºC; 2) LD/8ºC animals (n = 20)
we e also housed in long days, bu he ambien em-
pe a u e o he colony oom was main ained a 8 ± 1ºC;
3) SD/20ºC animals (n = 20) we e housed in a sho -
day pho ope iod (L8:D16) wi h he colony oom em-
pe a u e se a 20 ± 1ºC; and 4) SD/8ºC, animals (n =
20) we e housed in sho days, bu in a oom wi h
empe a u e kep cons an a 8 ± 1ºC. Animals we e
main ained in hei espec i e condi ions o 6 weeks.
A his ime, animals in each o he ou expe imen al
g oups we e sub- di ided u he in o ood es ic ed
g oups o ad libidum ed g oups. Food es ic ed ani-
mals ecei ed 70% o hei o iginal baseline ood in ake
while ad libidum ed animals con inued o ha e ee
access o ood. Food es ic ion con inued o 4 weeks.
P ocedu e
A e 10 weeks, animals we e b ough in o he su -
ge y oom one a a ime and ligh ly anes he ized wi h
me hoxy lu ane apo s (Me o ane, Pi man-Moo e,
Mundelein, IL). Handling ime was kep consis en
and o a minimum; he ime om ini ial emo al om
he cage o he end o bleeding was less han 3 min.
Blood samples (500 µl) we e d awn in o capilla y
ubes ia e o-o bi al bleeding (Riley, 1960) be ween
1000 and 1200 h EST, a ime when co icos e one al-
ues ha e been shown o be consis en ly low (Taymans
e al., 1997). All samples we e allowed o clo o 1 h.
The clo was emo ed, and he samples we e cen i-
uged (a 4ºC) o 1 h a 3500 pm. Se um aliquo s we e
ex ac ed and s o ed a –80ºC un il assayed.
A e blood sampling, all animals we e killed by
ce ical disloca ion. Pai ed es es, epididymides,
seminal esicles, b own adipose issue (BAT), and
epididymal whi e adipose issue (EWAT) we e
emo ed and cleaned o connec i e issue. Spleens
we e emo ed unde asep ic condi ions and immedi-
a ely suspended in cul u e medium (RPMI). Seminal
esicles we e comp essed wi h a glass ial o emo e
seminal luid. All o gans we e weighed by labo a o y
assis an s nai e o he expe imen al hypo heses and
ea men assignmen s. Tissue masses we e co ec ed
o o al body mass, and bo h absolu e and ela i e
masses we e used in subsequen s a is ical analyses.
Splenocy e P oli e a ion
Splenocy e p oli e a ion in esponse o he T-cell
mi ogen Con A was de e mined using a colo -
me ic assay based on he e azolium sal 3-(4,5-
deme hyl hiazol-2-yl)-5-(3-ca boxyme hoxyphenyl 2-
(4-sul ophenyl)-2H- e azolium (MTS). Splenocy es
we e sepa a ed om issue by comp essing he whole
spleen be ween s e ile os ed glass slides; sepa a ed
cells we e suspended in 4 ml o cul u e medium
(RPMI-1640/Hepes supplemen ed wi h 1% penicillin
Demas, Nelson / IMMUNE FUNCTION IN DEER MICE 255
[5000 U/ml]/s ep omycin [5000 µl/ml], 1% L-
glu amine [2 mM], 0.1% 2-me cap oe hanol [5 2× 10–2
M], and 10% hea -inac i a ed e al bo ine se um).
Splenocy e coun s and iabili y we e de e mined wi h
a hemacy ome e and ypan blue exclusion. Viable
cells (which exceed 95%) we e adjus ed o 2 × 106
cells/ml by dilu ion wi h cul u e medium, and 50-µl
aliquo s o each cell suspension (i.e., 100,000 cells)
we e added o he wells o s e ile, la -bo om, 96-well
cul u e pla es. Con A (Sigma Chemical Co., S . Louis,
MO) was dilu ed wi h cul u e medium o concen a-
ions o 40, 20, 10, 5, 2.5, 1.25, and 0.60 µg/ml; 50 µl o
each mi ogen concen a ion was added o he wells o
he pla e con aining he spleen cell suspensions o
yield a inal olume o 100 µl/well (each in duplica e).
Pla es we e incuba ed a 37ºC wi h 5% CO2 o 48 h
p io o addi ion o 20 µl o MTS/phenazine me ho-
sulpha e solu ion (0.92 mg/ml o PMS in s e ile Dul-
becco’s phospha e-bu e ed saline; P omega, Madi-
son, WI) pe well. Pla es we e hen incuba ed a 37ºC
wi h 5% CO2 o an addi ional 4 h. The op ical densi y
(OD) o each well was de e mined wi h a mic opla e
eade (model 3550, Bio-Rad, Richmond, CA)
equipped wi h a 490-nm wa eleng h il e . Mean OD
alues o each se o duplica es we e used in sub-
sequen s a is ical analyses. Dose esponse cu es
we e cons uc ed using g oup means o he mean OD
alues a each mi ogen concen a ion and uns imu-
la ed cul u es.
Se um Co icos e one Assay
Blood se um co icos e one concen a ions we e as-
sayed by adioimmunoassay (RIA) using he ICN
Biomedicals Inc. (Cos a Mesa, CA) 125I ki . The dee
mouse dilu ion was p epa ed acco ding o he guide-
lines u nished by ICN. The co icos e one assay was
highly speci ic, c oss- eac ing a less han 3% wi h
o he s e oid ho mones. Se um co icos e one alues
we e de e mined in a single RIA. The coe icien s o
a iabili y we e all below 10, and in a-assay a ia ion
was less han 1%.
S a is ical Analyses
Rep oduc i e o gans and o he issue masses, as
well as se um co icos e one concen a ions, we e
analyzed using a 2 (pho ope iod) × 2 ( empe a u e)
× 2 ( ood a ailabili y) be ween-subjec s analysis o
a iance (ANOVA). Splenocy e p oli e a ion was ana-
lyzed using a 2 (pho ope iod) × 2 ( empe a u e) × 2
( ood a ailabili y) × 8 (mi ogen concen a ion) mixed-
model ANOVA. Signi ican in e ac ions we e p obed
using indi idual wo-way ANOVAs, and all pai wise
compa isons o mean di e ences we e conduc ed
using Tukey hones ly signi ican di e ence compa i-
sons. Di e ences be ween g oup means we e consid-
e ed s a is ically signi ican i p < .05.
RESULTS
Splenocy e p oli e a ion was signi ican ly ele a ed
in mice main ained in mild ambien empe a u es in
sho - compa ed o long-day dee mice a e aged
ac oss eeding condi ions (p < .05) (Fig. 1). Food-
es ic ed animals demons a ed educed splenocy e
p oli e a ion compa ed o ad libidum ed animals (p <
.05) (Fig. 1). Food es ic ion educed splenocy e p o-
li e a ion in long- bu no sho -day mice housed in
mild ambien empe a u es. Howe e , ood es ic ion
educed splenocy e p oli e a ion in bo h long- and
sho -day mice housed in low ambien empe a u es
(p < .05 in bo h cases) (Fig. 1).
Food es ic ion ele a ed co icos e one in sho -
day animals main ained in low bu no mild ambien
empe a u es compa ed o ad libidum ed mice (p <
.05) (Fig. 2). A simila end was p esen in long-day
animals; howe e , he esul s we e no s a is ically
signi ican (p > .05). Se um co icos e one concen a-
ions we e signi ican ly lowe in ad libidum ed mice
main ained in low empe a u es in sho compa ed o
long days (p < .05) (Fig. 2).
Food- es ic ed mice weighed signi ican ly less
han ad libidum ed animals (p < .05) (Fig. 3). Body
mass was no signi ican ly a ec ed by ei he pho o-
pe iod o empe a u e (p > .05). Pai ed es es masses
we e smalle in sho -day dee mice compa ed o long-
day animals (p < .05) (Fig. 4). Simila ly, pai ed es es
we e signi ican ly smalle in sho -day, ood- es ic ed
animals main ained in low empe a u es ela i e o
sho -day animals main ained in mild empe a u es
(p < .05) (Fig. 4). Long-day, ood- es ic ed animals had
signi ican ly smalle pai ed es es compa ed o ad
libidum ed animals (p < .05 in bo h cases) (Fig. 4).
Pai ed epididymides we e smalle in ood- e-
s ic ed compa ed o ad libidum ed mice main ained
in low ambien empe a u es in bo h long and sho
days (p < .05 in bo h cases) (Fig. 5). Pai ed
epididymides we e also signi ican ly smalle in ad
libidum ed mice main ained in low ambien empe a-
256 JOURNAL OF BIOLOGICAL RHYTHMS / June 1998
u es compa ed o ad libidum ed mice main ained in
mild empe a u es. EWAT was signi ican ly educed
in long-day, ood- es ic ed mice compa ed o long-
day mice ed ad libidum (p < .05) (Table 1). Food-
es ic ed mice had signi ican ly less BAT han ad
libidum ed animals when main ained in low ambien
empe a u es (p < .05) (Table 1). No o he s a is ically
signi ican di e ences we e obse ed.
Figu e 1. Mean (±
SEM) splenocy e p oli e a ion o concana alin A (Con A) (µg/ml) o ood- es ic ed o ad libidum ed male dee mice
housed in long (L16:D8) o sho (L8:D16) days and mild (20ºC) o low (8ºC) empe a u es. Splenocy e p oli e a ion is ep esen ed as
abso bance uni s. Columns wi h no symbol o sha ing he same symbol a e s a is ically equi alen . Columns wi h di e en symbols a e
signi ican ly di e en a p < .05. Only he op imal concen a ion o Con A (i.e., he concen a ion ha s imula ed he highes amoun o
cell p oli e a ion) is g aphically ep esen ed.
Figu e 2. Mean (±
SEM) co icos e one le els (ng/ml) o male dee mice housed in long (L16:D8) o sho (L8:D16) days and mild (20ºC)
o low (8ºC) empe a u es. Splenocy e p oli e a ion is ep esen ed as abso bance uni s. Columns wi h no symbol o sha ing he same
symbol a e s a is ically equi alen . Columns wi h di e en symbols a e signi ican ly di e en a p < .05. Only he op imal concen a ion
o concana alin A (i.e., he concen a ion ha s imula ed he highes amoun o cell p oli e a ion) is g aphically ep esen ed.
Demas, Nelson / IMMUNE FUNCTION IN DEER MICE 257
DISCUSSION
In gene al, ep oduc i e physiology was inhibi ed
and immune unc ion enhanced in male dee mice
main ained in sho compa ed o long days. The sup-
p essi e e ec s o ood es ic ion on ep oduc i e and
immune unc ion in long-day animals we e o e come
by main aining animals in sho days. Sho -day, ood-
es ic ed mice main ained in low ambien empe a-
u es demons a ed educed ep oduc i e and im-
Figu e 3. Mean (±
SEM) body mass (g) o ood- es ic ed o ad libidum ed male dee mice housed in long (L16:D8) o sho (L8:D16) days
and mild (20ºC) o low (8ºC) empe a u es.
Figu e 4. Mean (±
SEM) pai ed es es mass (mg) o ood- es ic ed o ad libidum ed male dee mice housed in long (L16:D8) o sho
(L8:D16) days and mild (20ºC) o low (8ºC) empe a u es. Splenocy e p oli e a ion is ep esen ed as abso bance uni s. Columns wi h no
symbol o sha ing he same symbol a e s a is ically equi alen . Columns wi h di e en symbols a e signi ican ly di e en a p < .05. Only
he op imal concen a ion o concana alin A (i.e., he concen a ion ha s imula ed he highes amoun o cell p oli e a ion) is g aphically
ep esen ed.
258 JOURNAL OF BIOLOGICAL RHYTHMS / June 1998
mune unc ion compa ed o ei he ad libidum ed mice
o ood- es ic ed mice main ained in mild ambien
empe a u es. Taken oge he , hese esul s sugges
ha ood es ic ion ac s as a s esso and supp esses
immune unc ion; main enance o mice in sho days
can o e come he immunosupp essi e e ec s o ood
es ic ion. Low ambien empe a u e alone did no
a ec immune unc ion; howe e , when low empe a-
u e was combined wi h ood es ic ion, immune
unc ion was comp omised in sho -day animals.
Unlike he e ec o ood es ic ion alone, educed
ep oduc i e and immune unc ion in long-day, ood-
es ic ed mice main ained in low ambien empe a-
u es was no coun e ac ed by main aining animals in
sho days. Co icos e one concen a ions we e gene -
ally ele a ed in ood- es ic ed mice compa ed o ad
libidum ed animals, suppo ing he hypo hesis ha
educed ood a ailabili y ac s as an en i onmen al
s esso . Also, main aining animals in sho days
appea ed o educe co icos e one concen a ions in
ad libidum ed mice housed in low ambien empe a-
u es. These esul s sugges ha pho ope iod, ambien
empe a u e, and ood a ailabili y in e ac o a ec
bo h ep oduc i e and immune unc ion in dee mice.
Table 1. Mean (± SEM), ep oduc i e o gan and a pad mass (mg) in ad libidum ed o ood es ic ed dee mice housed in long (L16:D8) o
sho (L8:D16) days in mild (20ºC) o low (8ºC) ambien empe a u es.
Tempe a u e Food Seminal Vesicles Epididymal Whi e Adipose Tissue B own Adipose Tissue
L16:D8 20ºC Ad libidum 85.6 ± 8.0 128.8 ± 77.0 150.3 ± 11.0
20ºC Res ic ed 71.5 ± 25.0 67.4 ± 10.0 130.4 ± 25.0
8ºC Ad libidum 102.1 ± 38.0 140.8 ± 36.0 128.3 ± 6.0
8ºC Res ic ed 69.8 ± 10.0 60.0 ± 7.0 110.7 ± 12.0
L8:D16 20ºC Ad libidum 21.4 ± 1.5* 80.4 ± 7.0* 154.4 ± 9.0
20ºC Res ic ed 57.7 ± 6.0 75.1 ± 23.0 147.6 ± 16.0
8ºC Ad libidum 47.4 ± 4.0 75.9 ± 6.0* 163.7 ± 8.0
8ºC Res ic ed 49.8 ± 9.0 71.7 ± 12.0 133.6 ± 14.0*
*indica es a s a is ically signi ican di e ence be ween pho ope iodic condi ions.
Figu e 5. Mean (±
SEM) pai ed epididymal mass (mg) o ood- es ic ed o ad libidum ed male dee mice housed in long (L16:D8) o
sho (L8:D16) days and mild (20ºC) o low (8ºC) empe a u es. Splenocy e p oli e a ion is ep esen ed as abso bance uni s. Columns wi h
no symbol o sha ing he same symbol a e s a is ically equi alen . Columns wi h di e en symbols a e signi ican ly di e en a p < .05.
Only he op imal concen a ion o concana alin A (i.e., he concen a ion ha s imula ed he highes amoun o cell p oli e a ion) is
g aphically ep esen ed.
Demas, Nelson / IMMUNE FUNCTION IN DEER MICE 259
The deg ee o gonadal eg ession obse ed in he
p esen s udy, al hough s a is ically signi ican , was
uncha ac e is ically small o his species. The lack o
ull gonadal eg ession likely explains why sho -day,
ood- es ic ed mice main ained in mild ambien em-
pe a u es did no ha e signi ican ly smalle pai ed
es es compa ed o long-day mice. Addi ionally, a
small popula ion o dee mice a e ep oduc i ely non-
esponsi e o pho ope iod (i.e., gonadal eg ession
does no occu in sho days). Al hough some o he
sho -day dee mice in he p esen expe imen
appea ed o be ep oduc i ely non esponsi e o pho-
ope iod, hese animals we e included in he s a is ical
analyses in o de o ensu e su icien s a is ical powe .
Also, no signi ican co ela ion exis ed be ween pai ed
es es size and splenocy e p oli e a ion, sugges ing
ha enhanced immune unc ion in sho days may no
be ela ed o he deg ee o gonadal eg ession wi hin
P. manicula us (c . Demas e al., 1997).
Animals equi e a balanced ene gy budge in which
ene gy a ailabili y equals ene ge ic expendi u es
(Sheldon and Ve huls , 1996). S ess ul condi ions
such as low ambien empe a u es and educed ood
a ailabili y p esen du ing win e ele a e glucoco i-
coid concen a ions (Bha naga e al., 1995; Mu phy
and Wideman, 1992). Win e en i onmen al s esso s
dec ease ene gy a ailabili y and simul aneously in-
c ease ene gy demands h ough an inc ease in me a-
bolic a e (Wunde , 1984). Rep oduc ion is ene ge i-
cally expensi e, and bo h ep oduc i e unc ion and
b eeding a e cu ailed du ing imes o educed ene gy
a ailabili y (B onson, 1989; Wade and Schneide ,
1992). Addi ionally, immuni y can be ene ge ically ex-
pensi e (Demas e al., 1997; Henken and B andsma,
1982); moun ing an immune esponse equi es using
esou ces ha could o he wise be alloca ed o o he
physiological p ocesses. Thus, i is likely ha immune
unc ion, like ep oduc ion, is educed du ing imes o
se e e ene ge ic sho age, especially win e .
The esul s o he p esen expe imen sugges ha
ood es ic ion, in addi ion o educed ambien em-
pe a u e (Demas and Nelson, 1996), can ac as an
en i onmen al s esso and supp esses bo h ep o-
duc i e and immune unc ion in dee mice. Addi ion-
ally, main aining animals in sho days can coun e ac
some, bu no all, o he s ess-media ed supp ession
o ep oduc ion and immuni y. The p esen esul s
appea o econcile p e ious indings o supp essed
immune unc ion in ield s udies o seasonal changes
in immuni y (Lochmille e al., 1994; Shi a che a and
Hadjiolo , 1987; Sidky e al., 1972) wi h sho -day
enhancemen o immune unc ion epo ed in labo a-
o y s udies o pho ope iodic changes in immuni y
(B aina d e al., 1987; Vaughan e al., 1987; V iend and
Laube , 1973). Al hough sho pho ope iods enhance
immune unc ion (Nelson and Demas, 1996), labo a-
o y s udies ha e ypically been conduc ed wi h ad
libi um access o ood and wa e , mild ambien em-
pe a u es, educed social in e ac ions, and lack o
p eda o y p essu es. The p esen inding ha ood
es ic ion educes splenocy e p oli e a ion sugges s
ha se e al en i onmen al ac o s media e seasonal
changes in ep oduc i e and immune unc ion in dee
mice in he wild. These esul s demons a e he impo -
ance o manipula ing mul iple a iables when exam-
ining seasonal changes in physiological o beha io al
p ocesses. T adi ionally, mos labo a o y s udies o
seasonali y ha e elied solely on manipula ions o
pho ope iod, and in many cases he esul s o hese
s udies con adic he esul s o ield s udies. The p es-
en esul s sugges ha a mul i ac o ial app oach o
he s udy o seasonali y wi hin he labo a o y can
yield mo e ecologically ele an esul s, as well as
econcile some o he appa en disc epancies be ween
labo a o y and ield da a.
In con as o ou p e ious indings (Demas and
Nelson, 1996), educed ambien empe a u e alone did
no impai immune unc ion in he p esen expe i-
men . This di e ence is likely due o di e ences in
immunological assessmen ; o al IgG p oduc ion was
assessed in he p e ious s udy, whe eas lymphocy e
p oli e a ion o Con A was assessed in he p esen
s udy. Fo example, i is possible ha humo al immu-
ni y is mo e sensi i e o changes in ambien empe a-
u e compa ed o cell-media ed immuni y, al hough
his possibili y emains o be es ed. Howe e ,
educed ambien empe a u es did appea o a ec
immune unc ion when combined wi h ood es ic-
ion. These esul s p o ide suppo o an in e ac i e
e ec o en i onmen al ac o s on immune unc ion.
In sum, he p esen da a sugges an impo an
physiological and adap i e unc ional ole o pho o-
pe iod-media ed enhancemen o immune unc ion.
The ne e ec o ele a ed immuni y in sho days
appea s o be o coun e ac he supp essi e e ec s o
en i onmen al s esso s such as low ambien empe a-
u es o educed ood a ailabili y. Fu he mo e, he
in e ac ion be ween en i onmen al ac o s and immu-
ni y mus be conside ed o unde s and seasonal adap-
a ions in he wild.
260 JOURNAL OF BIOLOGICAL RHYTHMS / June 1998
ACKNOWLEDGMENTS
The au ho s hank John Dunlop, Joyce Hai s on,
Viole e Rena d, and Sue Yang o echnical suppo ,
D . Cou ney DeV ies o help wi h he co icos e one
assay, and Ed Sil e man o expe animal ca e. We
also hank Lance K iegs eld, Debbie D azen, and
Sab a Klein o aluable commen s on he manusc ip .
This s udy was suppo ed by NICHD G an MH 57535
and NSF G an IBN 97-23420.
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