Res. Popul. Ecol. (1962) IV, 8 16

A D R E N A L A N A L Y S I S F O R C R I T 1 0 U E O F T H E S O C I A L

S T R E S S T H E O R Y IN N A T U R A L P O P U L A T I O N S

O F A M O N T A N E V O L E

R y o TANAKA

Zoological Laboratory, Kochi Women 's Universi ty , Kochi

The preceding studies (8~(9~ of the variation in adrenal weight due to breeding

activi ty in natural populations of the Norway rat has demonstrated that the adrenal

weight is enhanced decidedly with increasing fecundity in females but on the contrary

tends to be lowered in males, and the view was advanced that such sources of error

must be well excluded in an analytic method for study of the social stress.

The phenomenon is already widely perceived in females but still of a limited

knowledge in males, which alone then are often used as a sample for analysis on the

unsound supposition that their adrenals were free from the breeding effect. The reverse

correlation of male adrenal weights with breeding levels was found in Microtus (~

and in Sigmodon (6~ so far as I am aware of. MCKEEVER (5) offered a justification to

the fact in the light of endocrinological knowledges up to date. NEGUS c t a ' . (6~

claimed that the change in adrenal weight appears to be at least as much a reflection

of reproductive influences as an index to a sociopsychological-physiological feed-back

system.

The present study in the vole, Eoahenomys smithi, has evidenced that the adrenal

weight in both sexes is more obviously variable than in the Norway rat under the

influence of the fert i l i ty level as well as the growth in terms of body weight or size.

Therefore I come to conceive that probably the like occurs to all kinds of small

mammals in different degrees characteristic of each species. From this view-point,

most of the papers, thus far published, aiming at proofs or disproofs or the CaRISVlA'~'S

theory through the adrenal analysis would seem to be more or less defective in

methods, and some evidence afforded by CHRISTIAN himself would not escape from

the same criticism.

POPULATIONS AND METHODS FOR STUDY

The animals were collected in about ten localities over the central highlands of

Shikoku during Nov. 1958--Jan. 1960. For the purpose of the general census of small

mammals performed under the direction of Mr. T. ITO, Shikoku Branch of Government

Forest Experiment Station, they were caught with snap traps by stuffs of the local

Ranger Stations of the Kochi Government Forest Office. A brief report of the census

was published in 1960 (1~

The vole populations in this district showed an upswing trend altogether toward

the end of 1958 and were at a peak the following two years, and went on declining

after 1961 onward. The peak was far less notable than that of the 1955 outbreak.

Then the sample, taken mainly from the populations of the mild peak years (1959-

1960) is supposed to include members of populations at various stages of growth and

at different density levels, since the collection was made in different months of the

year.

Out of the original specimens numbering 600 or so, 207 males and 135 females were

available to the adrenal analysis, the sample size being enough for a statistical test.

All the specimens were sent to the ITo's laboratory after every 2cc formalin had been

injected into a corpse in each locality, and measured by him of body weight (BW),

head and body length (HB) and testis size. A rough record of the breeding status

of female sexual organs was noted by him. Further I removed adrenals from these

specimens and scrutinized ovaries and uteri to determine the fecundity level in females.

The BW-value, given by ITo, minus 2g was deemed as a true BW. The HB-value

of ITo, gained from corpses somewhat contracted by the fixative, was postulated to

be about 10% reduced in each animal as compared to normal measures obtained prior

to fixation, and it has proved to be a grossly consistent index to a true HB. For the

inspection, 90 animals which I collected in 1959 and 1960 from Mt. Tsurugi were used.

Their BW and HB values, I measured by normal methods, created a correlation figure

between them which is concordant with that between I ro ' s measures, exclusive of a

corresponding transfer of regression lines. In the text ensuing, BW designates the

corrected, true value but HB does the uncorrected, original one.

In males, BW was used as a basic measure for adrenal analysis because it under-

goes far less influence of the reproductive phase than in females, where HB holding

utterly independent of fecundity was adopted instead ; it was thought better to use

the net body weight (BW minus total weight of ovaries, uteri and/or litters) as done

in the previous study c9~, but it could not by an unavoidable reason.

ADRENAL WEIGHT VERSUS BREEDING ACTIVITY

Ma'es : - The index of breeding activity was determined by making use of ITo's

testis dimensions as follows :

Testis length • width (ram) Index of male fecundity . . . . . BW (g)

By the amount of the index, the male sample was clsssified into 4 groups, I (0. 1~

0. 5, ) II (0. 6~1.0) , III (1.1~1.5) and IV (1.6 or more) ; we may be assured that the

fecundity level shifts increasingly from the lowest (I) to the highest (IV).

The relative adrenal weight (Y), i. e , paired adrenal weight (rag) per unit BW

(g) plotted on BW (X) resulted in giving the following regression equation applied

to the range of subadult and adult voles 16 g or more in BW :

10

Y = 0. 6 1 8 - 0. 0129 X ( X = 25. 7 g)

b = - 0 . 0129_k0. 00219 (c lear ly s ignif icant , P < 0 . 001, d . f . =205)

In both sexes, the onset of sexual m a t u r i t y is supposed to be at some 20 g in B W

equivalent to some 90 m m in HB (100 m m in a t rue HB) in view of the ex te rna l and

in terna l condi t ion of sexual o rgans and by reference to Figs . 1 and 2.

Fig. 1.

x /

;=

"O

o9

~9

X X

X I

Oil: 0.7 @m-

| , o • 0 . 6 - -

• x O X X

X @ 0.5-- X x @ X • @

x X _ x O

0.3 X )<xK

0.2 X

o.1 I I I I I. 0 i0 15 2~ 25 30

Body weight (g)

X X

35

Regression of relative adrenal weight on body weight in male voles ; I~IV show the index of fecundity, on which refer to the text.

F r o m the s ign i f ican t ly nega t ive regress ion, i t has proved t ha t even the re la t ive

adrena l weight is sure ly decreased wi th g rowth , then a pe r t inen t cor rec t ion is requi red

when means of the measure are compared be tween g roups of d i f ferent indexes. In

general , a mean (Y~) or a g roup is to be revised as follows, if the mean B W (X~)

of the g roup is dev ia ted f rom the genera l mean B W ( X ) :

~ • [X-X~[ • when X ~ X

T h e mean re la t ive adrena l we igh t s t hus cor rec ted are shown in Tab le 1.

In the male N o r w a y ra t (9), a nega t ive regress ion of the same measure was of a

border- l ined s ignif icance ( P - O . 05, d. f. =60) , so as to indicate tha t the amoun t of

regress ion, if any, was ve ry small , whi le in the female ra t no regress ion t r e n d was

11

found at all, accordingly a correction for the regression was regarded as unnecessary.

Table 1. Mean relative adrenal weights (corrected for regression) of each reproductive group in subadult and adult males 16g or more in BW.

Reproductive group I II III IV

n (No. of animals) 57 i 46 68 29

Mean (rag) 0.34=t=0.018 i 0.28~0.0t7 0.27~:0.012 0.25~0.022 Mean BW (g) 21.5 27.7 27.7 26.5

Ferna;es : -The regression of the relative adrenal weight (Y), i. e., paired adrenal

weight (mg) per 10 mm HB against HB (ram) (X) is exhibited in Fig. 2. The

regression line applied to the subadult and adult voles 80 mm or more in HB was

counted as follows :

3.5

i.0

& t,~ 2.5

2.0

v~

~9

Fig. 2.

1 . 5 -

1.0-

X"

L

•

OK

@m

O ~

0 0

0

0 0

0 0 e~. o

0

�9 @

|

• o o o

. o o

• ~ O o O O 0 ',-.u 0

I ~ I t 1 { I ,_ I __ } 65 70 75 80 85 90 95 100 105

Head and body length (ram)

Regression oI relative adrenal weight on head and body length in female voles ; I ~ I V show different levels of fecundity, on which refer to the text.

Y=0. 0379X-1. 571 (X=89.2 mm)

b=0. 0379=~0. 0 1 2 5 (significant, 0. 01>P>0 . 001, d. f. =133)

Thus we see that the relative adrenal weight is enhanced with growth in subadult

and adult stages opposite to males, and a corresponding correction for the regression

must be made likewise.

12

By the reproduc t ive level, the female sample was d iv ided into 4 groups as fol lows :

I : juveniles and adul t s in nonact ive or s l igh t ly act ive fecundi ty ,

II : adul t s in obviously act ive fecundi ty ,

III : adul t s having enlarged uteri , assumed as post pa r tum or lac ta t ing ,

IV : in visible pregnancy .

The cr i te r ion of the f irst two was set on the deve lopmenta l degree of ovar ies and

uteri. The mean adrenal weight of these groups is shown in Table 2.

Table 2. Mean relative adrenal weights (corrected for regression) of reproductive groups in subadult and adult females 80 mm or more in HB.

- = : - - : ~ = = :

Reproductive group I II III, IV

n 54 51 30 Mean (mg) 1.27-0.088 2.14• 2.21-~0. 131

Mean HB (mm) 86.0 90.8 92.1

T h e mean re la t ive adrena l weights , a t t ended by the i r 95% fiducial l imits , in differ-

ent reproduc t ive groups of both sexes are i l lus t ra ted in Fig. 3. The adrenal weight line

runs downward in males, while does upward in females, side by side wi th increas ing levels

of fecundi ty . In males, no s ignif icant difference is found be tween the three groups

highest in t e rms of fe r t i l i ty level, which are all m a r k e d l y smaller , especia l ly III and

IV being s ign i f ican t ly so, than the lowest g roup (I). Thus the overal l percentage

decrease in adrenal weight in the process tha t the male popula t ion shif ts f rom the

lowest to the highest level of fecundi ty can work out at 26. 5%.

In females, l ikewise, the h igher three groups, in which III and IV were combined

for the mean count because of a smal le r sample size (8 out of 30) in III, show a

s t r ik ing , s ignif icant super io r i ty over the lowest ([), and a s l ight d i f ference be tween

Fig. 3.

o.40

~: 0.35 .-g

0.30

0.25

0.20

j I

:.50

~.00 ~"

1 . 5 0 ~

%

..00

Illustrating the relation between relative adrenal weights and levels (I to IV) of breeding activity in both sexes.

13

I[ and I I I + I V migh t be due to the ming l ing of some voles in invisible p r e g n a n c y into

II. The overal l pe rcen tage increase of adrena l we igh t in t he upswing process (I to

IV) of the female popula t ion works out at as large as 74%.

Then, a combined popula t ion wi th a 50% sex rat io being t aken into account , the

adrenal change m a y be e s t ima ted at about 50% increase on balance as a joint resul t

of male decrease and female increase in the r is ing process of b reed ing ac t iv i ty , while,

on the con t ra ry , in the fa l l ing process (IV to I) a cor responding amoun t of decrease

in adrena l weight m a y well be expected .

The amoun t of adrenal change in the vole is comparab le to tha t I have given

previous ly ~9~ to the N o r w a y ra t which showed a 34% increase in females and a 14%

ins ign i f ican t decrease in males, then work in g out at 20 to 30% increase in combined

sexes, in the fecundi ty -upswing process. In the vole, as cont ras ted , the increase in

combined sexes amount s to as large as 50% and the male decrease is of a s ign i f ican t

quant i ty . Fu r the rmore , even the re la t ive adrena l we igh t undergoes a s ign i f ican t effect

of g rowth in body we igh t or size, whereas such was not at alI or scarcely d iscerned

of the N o r w a y rat.

Eventua l ly , we have ascer ta ined tha t the adrenal we igh t is much more spec tacu la r ly

inf luenced by rise and fall of r ep roduc t iv i t y as well as by shif t of age s t ruc tures in

the vole popula t ion than in the rat .

ADRENAL WEIGHT AND POPULATION DENSITY

Not all the da ta f rom each loca l i ty were avai lable for census, the avai lab le ones

having been obta ined f rom 13 plOts shown in Tab le 3. The popula t ion was e s t ima ted

by the equa t ion Ca--(N-S,,_~)p as usaul and the dens i ty per ha was counted by the

Table 3. Mean relative adrenal weights (corrected for regression) in subadult and adult males 16g or more in BW and breeding levels of each density group.

Density group Low Medium High

No. 2 (July '59) No. 10 (Jan. %0)

Pregnancy rate @d') in adult females

Mean relative adrenal weight (rag)

(n) (Mean BW in g)

Census plot (Date)

Average population 40. 5 72.2 136.8 density (per ha)

0 (0/18) 18.2 (10/55) 27.5 (11/40)

Nos. l,3,4,8,11,12, : No.5 (Nov. '58) 13, 14 Nos. 7,9 (May, Dec. (July, Oct., Dec. '59)i '59)

0. 26 A= 0. 024 0.26~0.012 i 9.31i0.031

(22) @9) (39) (25.6) (26.5) (24.9)

Active breeding rate 46.8 (11/24) 57.5 (61,/106) 48.8 (20/41) (~/o) in adult males* '

*Animal number of breeding groups III and IV divided by number of adults 19g or more in BW.

14

aid of the mean home range (0. 14 acres). The censused populations were divided

into the low (~50) , the medium (50~100) and the high (~100) group by the density

value. The mean relative adrenal weight, corrected for regression due to growth by

the same means as mentioned above, was calculated f rom subadult and adult males

of each density group (Table 3).

The adrenal is largest in the high group but no significant difference is denoted

among these groups, and yet the male fertility level is highest in the medium one.

Notwithstanding, the pregnancy rate is manifestly enhanced with the increasing density

in contradiction to the CHRISTIAN'S theory.

Since the same density group is supposed to be composed of animals at different

stages of population growth, this analysis might be inappropriate to the study of

density-dependent social pressures in view of his theory ~2) suggesting that the same

numerical density not always shows the same intensity of social pressure which is

chiefly dependent on a difference of growth stage. Be that as it might, the present

result seems to offer no proof in support of his theory.

DIscussioN

The MCKEEVER'S adrenal analysis ~5) in the vole, Microtus montanus, brought out

a result in some degree agreeable with mine ; namely, in the relative adrenal weight,

fecundity-active females are significantly superior to nonactive ones, while a converse

relation is seen in males, and the growth regression of the measure is positive for

females, while negative for males at least in adult stages. Nevertheless, it is quite

incomprehensible to us that his pregnant voles have the smallest adrenal among females

of other breeding groups except one with perforate vaginae. He computed the relative

weight per body weight after only the stomach weight was subtracted from the body

weight ; instead, he should have used the net body weight such as I remarked before.

Besides, he appears not to have made a correction for the regression, he was aware of,

to compare between the adrenal means of high and low density groups.

CHITTY ~t~ disclosed in the vote, Microtus agrestis, that pregnant and lactating

females have a larger adrenal than nulliparous ones, but failed to show a reverse

relation between males in active and nonactive breeding. The standardized mean

adrenal weight, he adopted, is an absolute weight corrected for a growth regression

but not a relative value per body weight. She was impossible to interpret sufficiently

the facts that the relation of adrenal weight to population density was just inconsistent

with between the two populations in different localities and a highest value in adrenal

weight appeared in June. If the relative adrenal weight corrected for regression and

arranged for avoiding reproductive effects had been used for comparison, another

better explanation might have been presented.

From Fig. 1 of CHRmTIAN c~>, we see that the nines of male albino mice evoked

only a slight increment (some 8%) in adrenal weight, which change might have been

15

ensued from the decline of fer t i l i ty ; if any social pressure had exerted additional

effects, a greater hypertrophy would have been induced. Thus one of the experiments

with the like sample by SOUTHWmK et al. (7)resulted in producing no significant

increase even in relative adrenal weight, nor did any testis decrease, but their proof

that wounded mice have clearly larger adrenals than nonwounded is suggestive of an

important effect of interaction upon adrenal activity. In conclusion, he did not go

to disclaim the social theory at all.

Louclt (3) in experimental populations of Microtus pennsylvanicus verified that the

blood eosinophil level was significantly in negative correlation with the density level

in Pens B and C, whereas the fecundity level kept invariable on the whole, and yet he

further witnessed a trend for the nestling or adult mortali ty to rise with the advanced

densi ty; these facts cause us to apprehend that the adrenal activity was enhanced

directly through increased social pressures, being followed by rise of the mortal i ty

but not by fall of the fecundity in favor of my conception induced in the preceding

papers. Nevertheless, since the fecundity level was not compared between the pen

populations, his comparison of male relative adrenal weights appears to have offered

no convincing evidence for the theory. In his next study (4) for natural populations

of this vole, the reproductive .effect on the male adrenal was quite set at naught in

interpretation of the adrenal visicitude. These papers are unsatisfactory to us in the

method that no correction for regression was made for relative adrenal weights.

SUMMARY

The vole, Eothenomys smithi , in highlands of Shikoku was studied to disclose ups

and downs of adrenal activities under the influence of the breeding level in natural

populations. I t has proved that the vole's adrenal in both sexes may be much more

strongly by rise and fall of the fertility as well as by shift of the age structure in

populations than the rat 's. Therefore, as for perhaps all kinds of small mammals ,

we consider necessary to follow a pertinent procedure to avoid thoroughly these

sources of error in studies to draw any conclusion for or against the CHRISTIAN'S

theory. The present data analysed by such a method failed to afford any confirmative

proofs ; the fact that the pregnancy rate was increased in paralleU to the density is

against his view. However, never the whole content of the theory has been invalidated

by these results.

REFERENCES

( 1 ) CmTTY, H. (1961) Variations in the weight of the adrenal glands of the field vole, Microtus agrestis. ]. Endocrin., 22: 387~393.

(2) CrmmTiA~, J.J. (1957) A review of the endocrine responses in rats and mice to increasing population size including delayed effects on offspring. Nay. ivied. Res. Inst., Lect. & Rev. Ser., 57-2 : 443~462.

16

( 3 ) Lovcn, C.D. (1956) Adrenocort ical act ivi ty in relat ion to the densi ty and dynamics of

three confined populations of Microtus pennsylvanicus. Ecology, 37 : 701~713.

( 4 ) Lovc~, C.D. (1958) Adrenocort ical act ivi ty in two meadow vole populations. J. Mcrnrn.,

39 : 1 0 9 ~ 1 1 6 .

( 5 ) MOKnEVER, C. (1959) Effects of reproduct ive act ivi ty on the weight of adrenal glands in

Microtus montanus. Anat. Rec., 135: 1~5.

( 6 ) N ~ v s , N.C., E. GovL~) and R.K. CmPMAS (1961) Ecology of the rice rat , Oryzomys pelustris,

on Breton Island, Gulf of Mexico, with a cri t ique of the social s t ress theory. Tulane Stud.

Zool., 8: 95~123.

( 7 ) Sov~Hwic~, C.H. and P.B. Viv~As (1959) Effect of population densi ty on adrenal glands and

reproduct ive organs of CFW mice. Am. J. Physiol., 197 : 117~114.

( 8 ) TA~xA, R. (1960) Adrenal hype r t rophy evoked by both increased social pressure and

reproduct ive act iv i ty in ra t populations. ]ap. ]. Appl. Ent. Zool., 4 : 226~233.

( 9 ) T~S~KA, R. (1962) F u r t h e r assurance of the effect of reproduc t iv i ty on adrenal weights

re levant to the social s t ress theory in a natural population of Norway rats. Bull. Kochi

Worn. Univ., 10 : 7~13.

(10) TAnaKA, R. and T. I~o (1960) Populat ion dynamics and damages of small mammals in

Shikoku in the year of 1959. Hopporingyo, 12 :340~344 (In Japanese).