Maternal behaviour of undernourished mother rats

Anim. Behav.,1973,21, 613-619

MATERNAL BEHAVIOUR OF UNDERNOURISHED MOTHER RATS BY J. L. SMART & JEANNETTE PREECE

University of Manchester, Department of Child Health, Clinical Sciences Building, York Place, Manchester M13 OJJ Abstract. Undernourished mother rats were given daily meals throughout most or all of pregnancy and lactation, comprising about half the amount of a good quality diet taken by controls. Their daily pattern of nest occupation and desertion during lactation was quite different from that of controls. Retrieving tests were carried out at 4, 6, 8 and 10 days after parturition. Underfed mothers were less efficient than controls in retrieving their young on days 8 and 10, and were less likely to lick their young during the test period. They also spent much more time rearing on their hind legs. Among undernourished females the number of young retrieved was negatively correlated with duration of rearing. The significance of the differences in maternal behaviour for offspring development is discussed. in differences in the subsequent development of the offspring (Barnett & Burn 1967; Meier & Shutzman 1968). Hence it is important to know whether these factors are inherent in the early underfeeding situations. This paper reports the results of the first part of an investigation of the maternal behaviour of undernourished mother rats.

In the last decade there has been a great intensification of interest in the effects of early nutritional deprivation on the development of brain and behaviour (see Scrimshaw & Gordon 1968; Smart 1971). The vast majority of investigations have been on the rat, and very often the period of nutritional deprivation has included the suckling period. This timing has been guided by the finding that, in terms of physical growth, the rat's brain is most vulnerable to experimental insult in the suckling period (Dobbing 1968). Even relatively mild undernutrition, imposed during the suckling period only, both impairs and distorts subsequent brain growth (Dobbing & Smart 1973) and has a lasting effect on brain enzyme activity (Adlard, Dobbing & Smart 1973). Recently, concern has been expressed about possible effects of the nutritional deprivation procedures other than their strictly nutritional ones (Plaut 1970; Frafikovfi 1971; Dobbing & Smart 1973). Stunting of suckling rats has usually been accomplished by one of the following three methods: (i) undernutrition or malnutrition of the mother; (ii) restricting the time available for feeding from the mother by taking some members of the litter away from her for several hours a day; (iii) giving the mother an abnormally large foster litter (say fifteen to twenty young). A reduction in the amount of maternal care would confidently be predicted to result from procedures (ii) and (iii). The implications of procedure (i) are not so obvious, but it would not be altogether surprising if the mother's nutritional state influenced her propensity to behave maternally. It has been suggested that differences in the quantity and quality of maternal care may result

Methods The rats were of the black and white hooded Lister strain. All females had borne and reared one to three litters before these experiments. At all times they were housed in cages 40 • 24 • 20 cm high (North Kent Plastics, RB3 cages). These comprised an opaque plastic base l 1-era high and a stainless steel lid 9-cm high, one end of which was recessed to hold food pellets and water bottle. The females were housed three to a cage with a male, and the day of mating was determined by daily examination of vaginal lavages for the presence of sperm. Lighting of the animal rooms was on a 12 hr white light/ 12 hr red light cycle, switching from white to red at 12.00 hours. Experiment 1 Females were housed singly from the day on which they mated. Wood wool was provided as nesting material during the third week of pregnancy. Nests were almost always made at the end of the cage away from the food and water. Control females had free access to a good quality diet (Breeding Diet for Rats and Mice, Oxoid Ltd, Southwark Bridge Road, London S.E.1) at all times. Undernourished females received 10 g a day of the same food throughout pregnancy, 15 g a day in the first 613

614

ANIMAL

BEHAVIOUR,

week of lactation, 20 g in the second and 25 g in the third. This was about half of what the control females consumed at comparable stages in their breeding cycle. Pellets for the underfed mothers were placed on the floor of the cage each day at about 09.30 hours. Water was available ad libitum at all times. On the day of birth, litter size was standardized to eight young. Rats were observed three times a day, at 09.00, 13.00 and 17.00 hours, 5 days a week, between day 1 (the day after parturition) and day 20 of lactation. As far as possible each observation was instantaneous and made without disturbance. On each occasion a note was made of whether the mother was in or out of the nest. There were sixteen control and eight undernourished mothers.

Experiment 2 Both groups of females were treated as in experiment 1 except that they were not housed singly till day 7 of pregnancy, at which time the underfeeding of one group was begun. Litters were again reduced to eight young at birth. Retrieving tests were carried out on days 4, 6, 8 and 10 of lactation; that is, each female was tested four times. Testing was between 14.00 and 16.00 hours in the red phase of the rats' white light/red light cycle. However, the observations were made in dim white light in a room adjoining the animal room. Retrieving tests were done in each mother's home cage, but the familiar cage lid was replaced with one empty of food and water bottle to facilitate observation. The experimental routine was as follows. The mother was placed in a holding cage, the young removed as far as possible from the nest within the confines of the home cage and the mother replaced in the empty nest. Retrieving responses, licking of the young, nest-building and rearing on the hind legs were recorded. Rearing was recorded whenever the rat lifted both forepaws from the floor of the cage, except when this was to manipulate nest material or self-groom. Each test lasted 5 rain, or if all the young had not been retrieved to the nest in that time, for 10 min. Young which had not been retrieved within 10 min were left for the mother to retrieve in her own time. Twelve control and twelve undernourished mothers produced litters but one undernourished mother lost her whole litter after the retrieving test on day 4.

21,

3

All tests of significance are two-tailed except where stated otherwise.

Results Experiment 1 The proportions of mothers in their nests at the three times of day sampled are shown in Fig. 1. Though there were a total of sixteen control and eight undernourished mothers, they were observed on only 5 days out of every 7 and hence most points are based on rather fewer observations than the total possible number. Throughout the 20-day period a higher proportion of control mothers were in their nests at 09.00 hours on all days (P<0.002). At 13.00 hours the difference was still in the same direction but not as clear-cut (P<0.008). However, by 17.00 hours the direction of the difference had reyersed; relatively fewer control rats were now in their nests than undernourished (P<0.002, all sign tests). Evidently the cyclic feeding regime profoundly altered the daily rhythm of nest occupation and desertion.

Experiment 2 Underfeeding the mother rats was effective in stunting the growth of their young, to the extent that by 10 days of age their body weight was less than half that of control offspring (Table I). The timing of the observations was guided by the results of experiment 1. Tests were carried out between 14.00 and 16.00 hours, at which time similar proportions of control and undernourished mothers would be expected to be in the nest. This prediction was confirmed during experiment 2. Expressions of retrieving efficiency are complicated by differences in litter size between the groups. Mortality was higher among the offspring of control mothers, such that by day 4 of lactation their mean litter size was 6.4 young, compared with 7-8 young for underfed mothers. By day 10 the mean litter sizes were 6.1 and 7.7, respectively. Unfortunately, mean retrieving time per nestling cannot readily be used, because of the number of occasions in which there was no retrieving within 10 min (a quarter of all tests with underfed mothers). Expressing retrieving performance as the proportion of mothers retrieving all their young within 10 rain favours the control mothers, as they had fewer young to retrieve (Fig. 2). A significantly higher proportion of control than undernourished mothers had complete retrieving on days 8 and 10 (P<0.02 and <0.01 respectively,

SMART & PREECE: M A T E R N A L BEHAVIOUR OF U N D E R F E D RATS

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AT 09.00 hr

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AT 13.00hr

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Q,

AT 17.00hr

.~

5

10

15

20

DAYS POST PARTUM

Fig. 1. Percentage of control (C) and undernourished (UN) mothers which were in their nests at 09.00, 13.00 and 17.00 hours each day from the first to the twentieth day after parturition. Table I. Body Weight of Young Rats: Mean 4- SD

Body weights (g) at Group

No. of litters

4 days

than on the first two. Change in performance between days 4 and 10 was analysed. Decrease in the time to retrieve all the young or increase

10 days

Control

12

11.2 • 2.0

23.2 4- 3.2

Undernourished

11

7.9 4- 1.0

11.1 4- 2-3

Fisher test). Conversely, another measure of retrieving efficiency, the number of young retrieved within 10 min, favours the undernourished mothers, as they have a higher possible score. Nevertheless, they scored lower than the control rats on three of the four tests (Fig. 3) and significantly so on day 10 (P<0.02 Mann-Whitney U-test). Evidently the difference in retrieving performanee is more apparent on the last two tests

100'

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~>-~s0o

r

u

DAYS POST PARTUH

Fig. 2. Percentage of control (C) and undernourished (UN) mothers which had retrieved all their young to ~ e nest within I0 min of the start of the tests.

ANIMAL

616

BEHAVIOUR,

z o

N 3'

o >-

o.--

g

~

UN

;

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i

10

"~

DAYS POST PARTUH

Fig. 3. Mean number of young retrieved to the nest by control (C) and undernourished (UN3 mothers within 10 rain of the start of the tests. Vertical bars represent sE.

in the number of young retrieved were taken to be improvements. Retrieving performance improved in nine out of twelve controls and deteriorated in eight out of nine underfed mothers (P<0.02, Fisher test). Two undernourished mothers showed no change. g 400'

~

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s **~176

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6 8 DAYS POST PARTUH

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Fig. 4. Mean retrieving latency (s) for control (C) and undernourished (UN) mothers. Vertical bars represent sE.

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3

An important component of the difference in retrieving efficiency was the difference in retrieving latency (Fig. 4). Retrieving latency was the time which elapsed from the mother being placed on her empty nest to her picking up the first of her nestlings. Undernourished mothers were significantly less prompt than controls to start retrieving on days 8 and 10 (both P<0.02, Mann-Whitney U-test). Of the other recorded measures of maternal behaviour, nest building did not differ between the two groups, but there were clear differences in propensity to lick the young (Table II). A significantly higher proportion of control mothers licked their young on three of the four test days. On the present evidence it appears that the control and underfed mothers differed in those aspects of maternal behaviour which involved contact with the young. Rearing on the hind legs was one of the most conspicuous activities in the test situation, especially among the underfed mothers. They spent much more time engaged in this behaviour than did control rats (P<0.005 on each day, t-test), yet there was no difference in the frequency of rearing responses (Fig. 5). Correlations between time spent rearing and the number of young retrieved are much less satisfactory for the controls than for the underfed group because of lack of uniformity in litter size at time of testing. Hence little importance should be attached to correlations for the control group (Table III). Among undernourished mothers duration of rearing was negatively correlated with young retrieved on all days of testing.

Table II. Numbers of Rats Observed Nest-Building and Licking Young in the First 5 min of Retrieving Tests Group

day 4

day 6

day 8

day 10

12

12

12

12

8

7

4

6

11

11

11

11

No. nest-building

5

5

3

6

Fisher test: P <

NS

NS

NS

NS

Control

No. licking young

5

8

9

11

Undernourished

No. licking young

0

4

2

3

Fisher test: P <

0.05

0'02

0"01

Control

Measure No. of rats No. nest-building

Undernourished

No. of rats

NS

SMART & PREECE: M A T E R N A L BEHAVIOUR OF U N D E R F E D RATS

617

Table m . Correlations Between Number of Young Retrieved to the Nest in the First 5 min and Time Spent Rearing

Group Control Undernourished

Statistic

day 4

day 6

day 8

day 10

*rs tP<

--0'30 Ns

--0.13 Ns

+0.37 Ns

+0.07 Ns

ra P<

---0.55 0"05

---0.64 0"05

---0.63 0.05

--0'56 0"05

* r s = The Spearman rank correlation coefficient. "~One-tailed test.

ISO"

I00"

o=

~

SO"

20,

1 q~--,.--,- C o. . . . . . UN

I0o DAYS POSY PARTUI4

Fig. 5. Mean frequency of rearing on the hind legs and total time spent doing so by control (C) and undernourished (UN) mothers in the first 5 min of the tests. Vertical bars represent sE.

Discussion The altered daily rhythm of nest occupation and desertion reported in experiment 1 for the underfed mothers was probably due to the cyclic feeding regime. That most of these mothers were out of their nests at 09.00 hours, about 89 hr before meal time, may reflect increased activity in the period before they were fed. Rats kept on a 23 hr a day food deprivation regime are twelve times as active (measured photoelectrically) as controls in the hour before feeding (Weasner, Finger & Reid 1960). Likewise, rats given about half their normal daily food intake at intervals of 24 hr and observed on a time-sampling basis, show a marked pre-

feeding peak in activity (Belles 1963). It is possible that this hyperactivity is a function both of the animal's internal state of nutritional lack and of secondary reinforcing factors: association of time of day and, in the present study, the presence of the experimenter with food presentation. Belles' finding that the underfed rats continue to be more active than controls for some hours after receiving their meal parallels the difference reported here in proportions of mothers out of their nests at 13.00 hours. His conclusion that 'The effect of deprivation is p r i m a r i l y . . , to change the pattern of waking activity rather than to increase the amount of it', appears to be equally applicable here. The length of time spent by the underfed mothers rearing on their hind legs may have contributed to their poor retrieving performance in experiment 2. Certainly the negative correlations between number of young retrieved and time spent rearing support this assertion (Table III). At simplest, rearing may have interfered with maternal behaviour, just because the t w o activities were almost always mutually exclusive. Alternatively, these two effects of undernutrition, decreased retrieving efficiency and increased rearing duration, may not have been causally related but m a y each have been linked to some other common factor. Sometimes, rearing was accompanied by sniffing, sometimes by bar-biting and sometimes rats walked in bipedal fashion while supporting themselves against the bars of the cage with their forelegs. In general, the character o f the behaviour was consistent with L~tt's (1965) interpretation of rearing as an index of excitability or arousal. In certain situations, rearing is an exploratory response, and some of the rearing observed here may have been investigatory in nature. Yet exploratory rearing is unlikely to have been a major component of the whole, as neither the

618

ANIMAL

BEHAVIOUR,

total frequency nor duration | rearing declined with successive tests. Some findings by Campbell & Sheffield (1953) may be relevant to the important question of why the undernourished mothers spent more time rearing. They measured activity in circular tilting cages both before and during several days of complete food deprivation. Environmental conditions were constant throughout each day, except for a 10-rain period when the levels o f sound and illumination were altered. The rats showed no change in total daily activity during the period of starvation from that in the free-feeding phase, but they were much more active during the 10 min of stimulus qhange when they were starved. On this evidence, starvation does not instigate activity; it lowers the threshold for normal stimuli to activity. Such an altered threshold could be invoked to explain the difference in time spent rearing by the control and underfed mothers. Another possibility is that the prolonged rearing of the undernourished rats was related to secondary reinforcing factors associated with food presentation. However, this seems unlikely since the cues were inappropriate: time of day, phase of lighting, treatment of cage, mother and litter, as well as the rat's internal state were all wrong. Furthermore, there was no extinction of the rearing response over the four test days in the absence of primary reinforcement. The assumption has been made up to now that differences in maternal behaviour were a function of differences in the mother. However, this may be quite unwarranted. There is clear evidence that maternal care by rodents can be influenced by the condition of the young (Young 1965; Barnett & Burn 1967). Since, in the present experiments, all mothers were tested with their own young, the deficient maternal behaviour of the underfed rats may partly have been due to differences between the control and undernourished offspring. Such underfed young are stunted in bodily growth compared with controls (Table I), show deficient motor responses at 5 days of age (Adlard, Dobbing & Smart 1973) and retarded neurological development in the second and third weeks of life (Smart & Dobbing 1971). These differences may not influence the nestlings' effectiveness in evoking maternal care, bu~ others as yet undetected, may do so. For instance, the production of ultrasonic squeaks, thought to be important in eliciting retrieving

21,

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(Noirot 1966; Sewell 1968) may be impaired in underfed young. The findings reported here are in good agreement with those of Frafikov~t (1971) on the maternal behaviour of malnourished mother rats. Mothers fed a low protein, high carbohydrate or low protein, high fat diet are less efficient than controls in retrieving their young and gain a lower overall maternal behaviour score. Also, the low-protein groups indulge in more 'exploratory' behaviour, one component of which is rearing. Rats mothering large litters have not been subjected to retrieving tests, but information exists on their behaviour in relatively undisturbed conditions. When housed in two-compartment boxes, rats with litters of twelve spent less time in the same compartment as the young than rats with litters of four, especially in the second half of lactation (Grota & Ader 1969). These findings are complemented by the time-sampling observations of Frafikov~t (1972). Not only were mothers of large litters seen to be out of their nests more often than those of small litters, but their distribution of activities within or outside the nest also differed. Hence there is good evidence that these methods of growth retarding rats during the suckling period alter some aspects of motherinfant interaction. These changes may, of themselves, influence the development of behaviour. The results of experiment 1 indicate that a cyclic feeding routine may cause a shift in the daily rhythm of nest occupation and desertion and thus also in the timing of motherinfant interaction. The intriguing question arises of whether this might affect the ontogeny of endogenous rhythms in the offspring. Acknowledgments The research was supported by grants from the Medical Research Council, with additional financial assistance from the National Fund for Research into Crippling Diseases. We wish to thank Miss Susan Moon for her part in the time-sampling study and Dr John Dobbing for his helpful criticism of the manuscript. REFERENCES Adlard, B.P.F., Dobbing, J. & Smart, J. L. (1973). An alternative animal modelfor the full-term smallfor-dates human baby. Biol. Neonate, (In press). Barnett, S. A. & Burn, J. (1967). Early stimulationand maternal behaviour. Nature, Lond., 213, 150-152. Bolles, R. C. (1963). Effectof food deprivationupon the rat's behavior in its home cage. J. comp. physiol. Psychol., 56, 456--460.

SMART & PREECE: MATERNAL BEHAVIOUR OF UNDERFED RATS Campbell, B. A. & Sheffield, F. D. (1953). Relation of random activity to food deprivation. J. comp. physioL Psychol., 46, 320-322. Dobbing, J. (1968). Vulnerable periods in developing brain. In: Applied Neurochemistry (Ed. by A. N. Davison and J. Dobbing), pp. 287-316. Oxford: Blackwell. Dobbing, J. & Smart, J. L. (I973). Early undernutrition, brain development and behavior. In: Ethology and Development (Ed. by S. A. Barnet0, pp. 16-36. London: Spastics Society with Heinemann. Frafikov~i, S. (1971). Relationship between nutrition during lactation and maternal behaviour of rats. Activ. nerv. sup. (Praha), 13, 1-8. Frai~kovfi, S. (1972). Influence of nutrition and early experience on behaviour of rats. Biblthca nutr. diet., 17, 96-110. Grota, L. J. & Ader, R. (1969). Continuous recording of maternal behaviour in Rattus norvegicus. Anim. Behav., 17, 722-729. L~lt, J. (1965). The spontaneous exploratory reactions as a tool for psychopharmacological studies. A contribution towards a theory of contradictory results in psychopharmacology. Proc. 2nd. int. pharmac. Meeting, 1, 47-66. Meier, G. W. & Schutzman, L. H. (1968). Mother-infant interactions and experimental manipulation: confoundingor misidentification ? DevlPsychobioL 1, 141-145.

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Noirot, E. (1966). Ultra-sons et comportements maternels chez les petits rongeurs. Annls Sac. r. zool. Belg., 95, 47-56. Plaut, S. M. (1970). Studies of undernutrition in the young rat: methodological considerations. Devl Psychobiol., 3, 157-167. Scrimshaw, N. S. & Gordon, E., Eds. (1968). Matnutrition, Learning and Behavior. Cambridge, Mass.: M.I.T. Press. Sewell, G. D. (1968). Ultrasound in rodents. Nature, Lond., 217, 682-683. Smart, J. L. (1971). Long-lasting effects of early nutritional deprivation on the behaviour of rodents. Psychiat. NeuroL Neurochir. (Amst.), 74, 443--452. Smart, J. L. & Bobbing, J. (1971). Vulnerability of developing brain. II. Effects of early nutritional deprivation on reflex ontogeny and development of behaviour in the rat. Brain. Res., 28, 85-95. Weasner, M. H., Finger, F. W. & Reid, L. S. (1960). Activity changes under food deprivation as a function of recording device. J. comp. physioL PsychoL, 53, 471-474. Young, R. D. (1965). Influence of neonatal treatment on maternal behavior: a confounding variable. Psychan. Sci., 3, 295-296. (Received 16 September 1972; revised 10 January 1973; MS. number: 1177)