Cosmet. Toxicol. Vol. 3, pp. 193-198. Pergamon Press 1965. Printed in Great Britain

The Influence of Environment on Behaviour, with Special Reference to Reprotlaeti~n in Mice

HILDA BRUCE

Medical Research Council, Department of Experimental Medicine, Tennis Court Road, Cambridge, England

Environmental factors and reproduction In all animals successful reproduction is dependent upon delicately balanced internal

mechanisms which reflect both internal and external changes. Reproductive behaviour is therefore closely related to environment.

The light/dark ratio is of paramount importance to the many species of animals which exhibit specific breeding seasons. Even among species long domesticated in the laboratory, which breed all the year round such as rats, mice and hamsters, ovulation and the occurrence of heat are associated with particular light conditions. The time at which these events normally take place can be altered by changing the 24 hr light/dark cycle to which the animals are subjected.

For example, in the Chinese hamster the female is frequently aggressive. She may savage the male or even kill him within a few hours of successful copulation. These animals must therefore be hand-mated and carefully watched so that the male can be removed to safety once the act of mating has been accomplished. The inconvenience of having to do this in the middle of the night when ovulation and mating usually take place, needs no comment. But by keeping the animals under a system of reversed lighting so that the females come into heat during normal working hours, instead of in the middle of the night, colonies of Chinese hamsters have been successfully established and maintained under laboratory conditions (Yerganian, 1958; Dobbelaar, van der Gulden & van Gaalen, 1963).

In warm-blooded as well as in cold-blooded animals changes in environmental tempera- ture are followed by changes in behaviour; as among the wild mice living in cold stores studied by Laurie (1946) and the colonies of laboratory mice maintained at -4°C by Barnett (1961).

Population density can lead to behavioural disturbance and reproductive failure. As long ago as 1931 Crew & Mirskaia (1931) demonstrated the relative inefficiency of large breeding groups of mice by comparison with smaller groups. More recently Christian & Lemunyan (1958) recorded an impairment of lactation that was carried over into the second generation among mice crowded for 6 weeks. Calhoun (1962) has described a gradual breakdown of individual behaviour in addition to reproductive disturbance among rats which were allowed to breed freely under conditions of limited housing until the colony became over crowded. At the other end of the scale, if the pregnant female is housed alone conditions" must be acceptable to her. Eleftheriou, Bronson & Xarrow (1962) found that pregnancy failed in a large proportion of Deer mice (Peromyscus maniculus bairdii), if the female was isolated in a large box instead of in the usual type of small box. And the same is true of laboratory mice (Bruce, 1963b).

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194 H |LDA BRUCE

Quite apart from inadequate diet and deficiencies of specific nutrients mere shortage of food can be detrimental to the establishment of pregnancy. A fast of 24 hr started on day 3 of pregnancy (copulation on day 0) will prevent implantation in about 50 ~ of mice (Bruce, 1963a). If the fast is extended to 48 hr, pregnancy fails in all mice (McClure, 1959).

Noise too can become a stress-agent sufficiently intense to cause not only adrenal changes and changes in eosinophil levels but also in behaviour (Anthony, 1963). Unexpected failures of reproduction associated with periods of undue noise, such as neighbouring building operations, have been experienced in many animal houses. But the evidence for such an association is conflicting, and we often remain ignorant of the factors really respon- sible for disturbed behaviour, that will, of course, differ in different species. Our traditional laboratory animals are still too often taken for granted, but biological response may be modified by the social conditions of a test in unexpected ways. Mtihlbock (1958) found that the incidence of mammary cancer was different among mice housed singly or in groups, and the toxicity of many drugs is influenced by the colzditions of caging during the test (Chance & Mackintosh, 1962).

These few examples of how the environment can affect behaviour, serve to emphasize the impossibility of achieving a comprehensive review of the subject in a short space. Rather than make the attempt, this communication aims at concentrating on one aspect only, namely social effects within the species, insofar as they are reflected in the reproductive cycle.

Social effects and reproduction Social effects on reproduction are known among various domestic animals but most or"

the observations in this field, and most of the experiments to be described, have been made on mice.

According to tradition, the mouse has a regular oestrous cycle of 4-5 days in length from puberty until senescence. The cycle is interrupted during pregnancy and lactation and by pseudopregnancy. Failure of the coital stimulus to break the oestrous rhythm by pregnancy (or by pseudopregnancy) is rare; among vigorous mice the spontaneous return of oestrus within 4-5 days of copulation will represent less than 10~ of matings. This picture is in- complete and even misleading for, except in the presence of the male, the oestrous cycle of the mouse is extremely flexible and reflects the social conditions under which the female is living.

When female mice are housed individually the cestrous cycle is from 5-6 days in length and pseudopregnancies are rare. By contrast when females are housed in small groups there is mutual suppression of oestrous and the incidence of spontaneous pseudopregnancy may amount to about 25 °/o of all cycles measured (Van der Lee & Boot, 1955). On the basis of a 4-5 day oestrous cycle, about equal numbers of females would be expected to mate during each of the first 4 or 5 nights after pairing but this does not occur unless the females have. been maintained individually before the introduction of the male. When grouped females are paired monogamously with stud males, far fewer than expected mate on the first and second nights after pairing, but matings over the third night greatly exceed expectation The reason for the peak mating is that when female mice are housed together in large numbers many become anoestrous; the introduction of the male initiates a new cycle so that oestrus is synchronized.

Not only is the anoestrus induced by the presence of other females terminated by the introduction of the male, but the oestrous cycle itself is shorter and more regular than when

I N F L U E N C E OF E N V I R O N M E N T O N M O U S E R E P R O D U C T I O N 195

the female is housed alone (Whitten, 1956, 1957, 1959). All these effects are attributed to olfactory stimuli (Van der Lee & Boot, 1956; Whitten, 1956). A simple experiment illus- trates these reactions by demonstrating the effect on the oestrous cycle of two changes in the olfactory environment of 30 female mice housed together since weaning, (1) the introduction of males and (2) their subsequent removal. Female mice that have been reared together since before puberty do not exhibit mutual disturbances of the oestrous cycle (Lamond, 1959). The reaction occurs essentially between strangers. Nevertheless the introduction of four males, caged individually to prevent contact, into the box containing the females, caused a significant increase in the incidence of oestrus. The subsequent removal of the males was also followed by an immediate reaction. Group identity had been destroyed, the females had become strangers to one another and oestrus was mutually suppressed, although the group was still composed of the same individuals and the conditions of housing were unchanged (Bruce, 1962b; Fig. 1).

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,oH OEST~US 4~TmM~s OESTRUS ~aT~MES OESTRUS 3 2 T I ~ S

FIG. I. Soc ia l e f fec ts o n oes t rus in m ice . i , o e s t r u s = v a g i n a | s m e a r f u l l y ¢ o m i f i e d .

These are all social responses of the unmated female, but equally impressive reactions to changes in the social environment are shown by the recently-mated female. If she is exposed to a male other than the stud male, particularly if the second male is alien to the stud male (i.e. belongs to a different strain) pregnancy is blocked so that oestrus returns automatically 4--5 days after the stud mating. If the second male has access to the female a fertile mating may take place but no superfoetation occurs, the first pregnancy has already been blocked so that only young sired by the second male are born. Sexual vigour does not contribute to the reaction; males completely lacking in sex drive may be effective pregnancy-blocking agents. Exposure to other females does not interfere with pregnancy (Bruce, 1959, 1960). As with social effects on the oestrous cycle, contact between the sexes is not necessary--proximity is sufficient, but unlike social effects on oestrus where maximum results are obtained within the strain, in pregnancy-block strain differences are important. Two sexes and 3 indi- viduals are concerned; there are therefore 5 ways in which strain differences can find expression. The reaction can take place (1) within a single strain, (2) between 2 strains, when the female belongs either to the same strain as the stud male, or to the same strain as the

196 HILDA BRUCE

second male or the 2 males belong to the same strain and the female differs from both, and (3) between 3 strains, each individual then being alien to the 2 others.

The combined results of tests involving several different strains (randomly-bred or in- bred) soon showed that a high proportion of blocked pregnancies could be expected when- ever the 2 males belonged to different strains. The strain to which the female belonged was not important nor did the introduction of a third strain into the reaction increase the pro- portion of blocked pregnancies (Parkes & Bruce, 1961). The behaviour of the female is determined by the identity of the stud male and by the extent to which she can perceive a difference between the 2 males.

The female is sensitive to this influence of males from the day of mating until after implantation, which in the mouse starts between days 4 and 5. By day 5 considerable resistance to male influence has developed and by day 6 the reaction is virtually abolished (Bruce, 1961).'The operative stimulus initiating the effect was found to be the smell of the male. Females rendered anosmic by surgical removal of the olfactory lobes were virtually immune to exposure to alien males (Bruce & Parrott, 1961).

Early attempts to separate the smell from the males were not successful, until a clue to the difficulty was given by the adoption of a new technique. If the newly mated female was housed in a box recently vacated by males, and the box changed twice dally so that she followed a group of males through a series of 6 boxes during the first 3 days after mating, pregnancy was blocked in about the same proportion of females as by the live male. Merely changing her into a clean box twice daily was without effect on pregnancy (Parkes & Bruce, 1962). The significance of this result lies in the fact that the smell had been separated from the male in sufficient concentration to block pregnancy in a high proportion of females. The immediate cause of the failure to implant brought about by olfactory stimuli of male origin is a lack of pituitary luteotrophin for if the female is given exogenous pro- lactin (10 I.U./g/day) during the 3 da2cs of test, her litter is carried to term in spite of expo- sure to males (Bruce & Parkes, 1961). Continued suppression of pituitary luteotrophic activity after successive matings, by olfactory stimulation of male origin, did not impair the subsequent fertility of the female, nor, as far as could be detected, was her sensitivity altered by repeated exposures (Bruce, 1962a).

Under natural conditions, isolation of the pregnant female would be rare. Exposure of the female to groups of males was without effect on the incidence of pregnancy block, at least up to groups of 8 males, the effect of a single male received no reinforcement from the presence of other males. By contrast the grouping of females together before exposure to the male, gave a marked degree of protection to pregnancy (Bruce, 1963b).

A tendency for females belonging to colonies of wild mice to congregate together has been noted by Davis (1958). Similarly Crowcroft (1962) reported that pregnant females tended to drive away other mice. Dr. J. Godfrey (personal communication) of the MRC Radiobio- logical Research Unit at Harwell indicated that among experimental colonies of laboratory mice such behaviour is also common.

Both the increase in the number of spontaneous pseudopregnancies when females are grouped together in the absence of males and the decrease in the incidence of the olfactory block to implantation when pregnant females are grouped before exposure to a male, are compatible with the suggestion that olfactory stimulation from other females, far from suppressing luteotrophic activity, as does olfactory stimulation of male origin, has a stimu- lating effect on the secretion of pituitary prolactin. The pathways by which these remarkable effects are achieved are still unknown. Presumably the hypothalamus is involved at some stage.

INFLUENCE OF ENVIRONMENT ON MOUSE REPRODUCTION 197

The olfactory-block to pregnancy has been demonstrated in the Deer mouse, Peromyscus (Eleftheriou et al. 1962) and social effects on the oestrous cycle are known to take place in voles Microtus agrestis (Chitty & Austin, 1957). The olfactory block to pregnancy has not been demonstrated in the laboratory rat, but Davis (1960) reported a temporary set-back in population growth among wild Norway rats in Baltimore following the introduction of "equal numbers of rats into increasing populations". His description suggests pregnancy- block in operation under natural conditions. Failure to produce the reaction under the laboratory conditions in which it occurs in mice, does not exclude the possibility of its existence in rats. Moreover, recently Everett (1963) has drawn attention to the importance of olfactory stimuli in the rat and to the effect of isolation on pseudopregnancy. Rats and mice are too often tacitly assumed to be similar. In fact they differ in many respects and their social responses to other members of their respective species may well come into this category.

Olfactory effects on the reproductive cycle are also recognized among domestic animals. Sheep (Schinckel, 1954) and goats (Shelton, 1960) can be brought into heat earlier in the season by the introduction of the male. The anoestrous ewe can be made attractive to the ram by annointing her with a swab taken from an oestrous ewe (Kelley, 1937). Sows will respond to the smell of the boar by the immobilizing reflex associated with copulatory behaviour (Signoret & du Buisson, 1961).

Fascinating as these social effects on reproduction are for themselves, the importance lies in their general implication for they reveal the activity in mammals of a most interesting group of substances only recently recognized and so far primarily associated with insects. The chemical messengers, with which we have long been familiar, "hormones" as originally defined by Starling, are substances produced by the internally-secreting glands for the integration of the individual. These new substances which can also be described as chemical messengers, are produced by externally secreting glands for communication within popula- tions. For chemical messengers of this type, i.e. between individuals, the term "pheromone" from two Greek words "pherein" to carry and "hormon" to excite or to stimulate has been proposed by Karlson & Butenandt (1959). Their work was concerned with the sex attrac- tants of insects, in particular the silkworm moth (Bombyx mori L.). The essential and dis- tinguishing characteristic of a pheromone is that it is produced by one individual to affect others of the same species. Substances such as the perfume of flowers which serve to attract insects are therefore not pheromones although they can also be described in general terms as chemical messengers. Pheromones are not necessarily olfactory, orally acting phero- mones, for example the queen substance of honey bees (9-ketodecenoic acid), are already known. This substance is secreted by the mandibular glands of the queen, and when in- gested by worker bees, inhibits the development of their ovaries and their ability to manufac- ture the queen cells in which new queens would be reared.

The scientific study of animal behaviour both in the wild and under laboratory condi- tions is a comparatively new discipline, more especially in the field of communication by means of which the environment in the broadest sense, affects the individual. We know little enough about communication between individuals belonging to the same species, and of the impzct of different species upon one another, still less.

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