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Effects of litter size on the behaviour of lactating female mice (Mus musculus)
Anita. Behav., 1972, 20, 386--394
EFFECTS OF LITTER SIZE ON THE BEHAVIOUR OF LACTATING FEMALE MICE
(MUS MUSCULUS)
BY ROBIN PRIESTNALL* Department of Psychology, The University, Leicester LE1 7RH Abstract. The behaviour of multiparous female C3H mice rearing different sized litters was examined using an observational time-sampling procedure, The results indicate that while animals rearing smaller litters spend more time in the nest, more time licking individual pups, and during most of lactation, more time nursing the litter, animals rearing larger litters spend more time eating and drinking. It is suggested that the observed differences in behaviour may be the outcome of the differential nutritional requirements arising from feeding litters of different sizes. However, in a second experiment providing food and water inside the nest, the differences in nest-attendance and nursing behaviour persisted, suggesting that some aversive factor such as fatigue or discomfort may also be involved. the time a lactating female rat spends with its litter can be taken as a reflection of maternal behaviour and some support to the validity of this assumption was obtained using a modified version of the maternal behaviour scale devised by Seitz (1958). The present study was designed to investigate both maternal behaviour and behaviour not directly concerned with the care of the young, in mice Mus musculus rearing litters of different sizes. The observational time-sampling technique employed involved no disturbance of either the mother or the offspring, while allowing the separate recording of many aspects of maternal behaviour; a procedure which would seem necessary in view of the low intercorrelations demonstrated between different measures of maternal behaviour (Slotnick 1967) and the general difficulties associated with the unitary drive concept (Hinde 1959, 1970).
The maternal behaviour of rats and, consequently, the early experience of the pups, has been shown to vary with the size of the litter. Seitz (1954, 1958), rating lactating female Wistar rats on nine observational categories, compounded a 'total maternal behaviour score' by addition of the ratings and found that mothers with smaller litters 'behaved significantly more maternally' than mothers with large litters. Seitz's conclusions which are based upon a parametric analysis of the rating-scale data assume implicitly that maternal behaviour is the outcome of a unitary drive. Draper (1968), working with thirteen fitters of primiparous Wistar female rats found that animals caring for smaller litters built more covered nests, while females with larger litters were more likely to move pups away from the nest during tests of retrieving. In this study, females were allowed to rear their own pups and the test procedure, like that of Seitz, involved considerable disturbance of both mothers and offspring. A later investigation by Grota & Ader (1969) employed a dual-chambered nesting cage which recorded electronically the time spent by Charles-River (CD) female rats with the litter in the compartment containing the nest, or away from the litter in the other compartment. The authors report that females caring for both large and small litters spent approximately 85 per cent of their time with the pups immediately after parturition, but the proportion of the time spent with the pups decreased more rapidly over the lactation period in females rearing larger litters. Grota & Ader (1969) assurded that *Present address: Department of Psychology,University of Sheffield,SheffieldSt0 2TN.
Experiment 1 Methods Subjects. The subjects were fifty-eight C3H female mice aged between 16 and 24 weeks at the beginning of the experiment. They had reared their first litters undisturbed and in isolation and were kept in all-female groups of ten to twelve animals for a period of at least 2 weeks before pairing in an attempt to induce oestrous synchrony (Whitten 1958, 1959). Holding conditions. All females were paired simultaneously with single males in small plastic laboratory cages (35 • 15 • 13era), and as soon as it became obvious that they were pregnant (usually during the second week of gestation), the females were transferred to specially constructed observation cages. The 386
PRIESTNALL: LITTER SIZE AND BEHAVIOUR IN THE MOUSE final transfer occurred at least 1 week before parturition and the females were then left undisturbed until the weaning of the litter, except for the manipulation of litter size immediately following parturition. The observation cages were 30 • 25 x 30 cm. The rear and side walls were constructed of 20 gauge galvanized mild steel sheet and the front wall was of Plexiglass. Food was supplied in a 75-mm mesh hopper on the rear wall of the cage and water in a plastic feeder inserted through the Plexiglass observation panel. The floor covering was sawdust and wood shavings, and each cage was supplied with 3 to 4 g of cotton wool for the construction of the nest. Animals were maintained on a diet of 41B animal food pellets. The whole experiment was carried out on a reversed light schedule; the room being in darkness from 09.30 to 19.30 hours. A series of four 100-W red lamps continually burning above the observation cages provided sufficient light for observation during the dark phase. Manipulation of litter size. As parturition became imminent, females were checked at least once every 12 hr and as soon as the litters were born the female and the litter were gently removed from the cage using padded forceps. The litter was placed in a tray of cotton wool heated from below by a 60-W lamp to avoid cooling while the remaining pups were collected. Synchronization of parturition was not totally successful but where two or more litters were present on any check period, the pups were pooled and then both females and pups allocated at random to the three experimental litter-size conditions of two, five and eight pups per litter. Allocation was sometimes limited by the number of pups available. The newly assembled litter was introduced into the nest of its assigned mother and finally, the female was returned to the corner opposite the nest in her own cage. Of the twenty litters of two pups made up in this way, sixteen were reared successfully to weaning. Nine of the fifteen litters of five pups and only three of the .twenty-three litters of eight pups were weaned intact. If one or more pups died during lactation the litter was usually discarded but, because of the high wastage of litters of five and eight pups, some litters suffering only one mortality were retained to increase the sample-size. Three litters of five that were reduced to four pups and four litters of eight reduced to seven were included in this way. A control experiment by the author (Priestnall 1970) revealed that this fostering procedure had
387
no significant influence upon any of the forms of behaviour recorded in this study. Observation of maternal behaviour. Observation was carried out on alternate days over the first 3 weeks of lactation. Females were observed on days 1, 3, 5, 7, etc., or on days 2, 4, 6, 8, etc., depending upon the day of birth of their litters. During the 4-hr observation period, sixty observations were made upon each of the females at intervals of approximately 4 min. This interval allowed sufficient time for observations on up to twenty-five cages to be recorded. During each 4-min cycle the experimenter moved from cage to cage noting on a prepared record sheet the form of behaviour displayed by each female at the moment of observation. The record was made in coded form, the following forms of behaviour being noted: In nest: The female was anywhere inside the nest, indulging in any form of behaviour at the moment of observation. Nursing: The female was allowing the pups to suckle. Nursing did not necessarily imply that the whole litter was nursing, nor that the female was adopting the nursing posture with her body arched over the pups. Licking pups: The female was licking or grooming her pups, Nest building: The female was engaged in some aspect of nest building behaviour at the moment of observation. She could be inside or outside the nest, pushing and kneading material into position with her snout and forepaws, shaping the nest, or pulling nest material towards it. Eating: The female was nibbling at a food pellet. Drinking: The female was drinking from the water container. Grooming: The female was grooming her own body. Resting: The female was lying motionless, either inside or outside the nest, usually with her eyes open but not involved in any other form of behaviour. Active: The female was moving about the cage at the moment of observation. The position of the young (inside or outside the nest) was also recorded together with any form of behaviour not catered for by the code. The next cycle of observation was not commenced until the 4-min interval had elapsed. The whole procedure was then repeated.
388
ANIMAL
BEHAVIOUR,
20,
Results
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From the record sheets, scores were obtained for each of the classes of behaviour, for each female, on each of the 4-hr observation periods by simply adding the number of observations in which that particular behaviour occurred. Any animal can therefore obtain a score of up to sixty on any class of behaviour, on each day of testing. Scores over the lactation period for each of the forms of behaviour recorded are presented graphically in Figs 1 to 9. The graphs are plotted from the mean scores for each experimental group on alternate days of lactation.
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Data for those classes of behaviour having a high incidence (in nest, nursing, eating, grooming and active) were analysed over the lactation period using an analysis of variance design with repeated measures on one factor. Because of the possibility that the differential mortality observed in different sized litters was related to litter size itself, the least squares solution (Winer 1962) was employed. The results of these analyses are summarized in Table I. For those classes of behaviour having a relatively low incidence (licking pups, licking per pup, nest-building, drinking and resting), total scores were calculated by adding scores over the ten observation periods. The totals were then treated by a single factor analysis of variance and the results of these analyses together with
PRIESTNALL: LITTER. SIZE AND BEHAVIOUR. IN THE MOUSE
389
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the mean total scores and the standard deviations are also available in Table I. Maternal behaviour. Most maternal behaviour occurs inside the nest; at least until the litter becomes active around 2 weeks post-partum. In all litter-size groups nest attendance declined steadily during the first 2 weeks of lactation, showing a slight increase during the third week. This increase was almost certainly due to the progressive flattening and consequent increase in area of the nest, together with the gradual loss of identity of the nest site as distinguished from the
Fig. 8. Resting. rest of the cage. Throughout the entire lactation period females rearing smaller litters were observed more frequently inside the nest. The behaviour most frequently observed in the nest was nursing. Analysis of the nursing scores revealed a highly significant interaction effect. During the first 2 weeks of lactation, females rearing smaller litters obtained the highest nursing scores, but during the third week, females rearing larger litters obtained the higher scores. This interaction effect might be accounted for in terms of a change occurring over the period of lactation in the mother-infant relationship
390
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Fig. 9. Active. with regard to the initiation of nursing. Such a change has been described for rats by R o s e n b l a t t & L e h r m a n (1963) a n d for mice b y Priestnall (1970). The end of the 2nd week of lactation is a c c o m p a n i e d b y o p e n i n g o f the p u p ' s eyes, a n
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increase in m o v e m e n t s by the pups away from the nest, a n d a decrease in the retrieval behaviour of the mother. A t this stage the pups begin to take a more active role i n the initiation of nursing, which they a t t e m p t b o t h inside a n d outside the nest. Pups from larger litters apparently o b t a i n less n o u r i s h m e n t ; they are lighter in weight t h r o u g h o u t the period o f lactation, a n d as a result of limitations o n the female's supply of time a n d milk they are p r o b a b l y rarely satiated. As the onus for initiating n u r s i n g passes to the pups, animals from larger litters may a t t e m p t to do so more frequently t h a n animals from smaller litters, a n d as the pups often solicit n u r s i n g individually outside the nest, they will tend to nurse at different times, with the result that females rearing larger litters will be observed n u r s i n g more frequently at this stage of development. N o significant difference was f o u n d i n the n u m b e r of observations i n which females rearing different sized litters were licking their pups b u t as only one p u p is licked at a time, the p u p ' s experience of licking is better represented by dividing the total licking score by litter size. A n analysis of variance executed after this transf o r m a t i o n indicated that pups reared in smaller litters receive significantly more licking from their mothers.
Table I. Analysis of Behaviour of Females Rearing Litters of Different Size Over the First 3 Weeks of Lactation
Mean total scores Behaviour
Litter size 2
Litter size 5
Litter size 8
Results of analysis of variance Litter size effect
380.9 336'3 261.6 F = 13'69, df = 2/32 (55.4) (40"3) (57.3) P < 0.005 Nursing 273"4 2 9 0 " 6 248.6 F = 20.44, df= 2/32, (51"3) (35"8) (43.9) P < 0.005 Licking pups 13"5 21'7 12-0 F = 2'20, df= 2/32, (4"9) (6"6) (5.3) Ns Licking per pup 6-8 4"3 1.5 F = 19.65, df = 2]32, (1"9) (1"4) (0'2) P = 0.005 Nest-building 9"8 20"3 16-4 F = 4"82, df = 2/32, (8-0) (7"6) (7.9) P < 0.05 Eating 126.8 142"0 224.0 F = 30.53, df = 2/32, (31'6) (20"6) (29.7) P < 0.005 Drinking 8.0 11"3 43.0 F = 11-46, df= 2/32, (3.5) (3"5) (32-3) P < 0.005 Grooming 96"2 82'8 69.0 F = 7.40, df = 2/32, (17.7) (19"8) (14.5) P < 0"005 Resting 54.4 18"3 10.7 F = 20.12, df= 2/32, (24.6) (9.8) (9-5) P < 0.005 Active 48.4 42'3 31-6 F ~ 1'39, df ~ 2/32, (28.4) (13"2) (17.7) NS The numbers in parentheses are standard deviations.
In nest
Days e f f e c t
Interaction effect
F = 29.85, df = 9/288 F = 1.36, df = 18/288 P < 0.005 Ns F = 49.28, df= 9/288, F = 496.51, df= 181288, P < 0.005 P < 0.005 ----
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F = 37.03, df = 9/288, F = 2.70, df= 181288, P < 0.005 P < 0.005 --F = 2.41, df = 9/288, P < 0.05 --
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F = 3.62, df = 9/288, P < 0.005
F -- 1.93, df= 181288, P < 0"05
PRIESTNALL: LITTER SIZE AND BEHAVIOUR IN THE MOUSE The statistical analysis suggests that females caring for the intermediate sized litters of five pups spend the most time involved in building and repairing the nest ( F = 4-82, df= 2/32, P < 0.05). However, the relatively low incidence of this form of behaviour and the high standard deviations of the mean total scores reduce the reliability of this finding. Nest building declined steadily in incidence over lactation, disappearing almost completely in all groups by the end of the second week post-partum. Non-maternal behaviour. The most frequently observed form of behaviour outside the nest was eating. Eating appeared to reach peak incidence in females in the large (litter size 8) and small (litter size 2) groups about 14 days after the birth of the litter. In the intermediate (litter size 5) group peak incidence occurred a little later, probably accounting for the significant interaction effect on this measure. The decline in food intake during the third week of lactation is probably related to changes in the rate of growth of the pups and to the transition of the litter onto solid food at this stage. Throughout lactation animals rearing larger litters were observed eating more frequently than animals rearing smaller litters, and drinking behaviour though much lower in incidence, showed a similar pattern; animals with larger litters being observed drinking significantly more often. On the other forms of non-maternal behaviour recorded; grooming, resting, and activity, females rearing smaller litters obtained higher scores, the differences in the grooming and resting scores being significant. A significant interaction effect was obtained on the activity score ( F - - 1.93, df= 18/288, P < 0.05) but in view of the low incidence of this class of behaviour and the great variability in the scores, this finding may not be reliable.
Experiment 2 The first experiment suggests that females caring for larger litters leave the nest more often, and consequently show less maternal behaviour, because of their greater need for food and water. Another possibility is that some aversive factor such as fatigue or discomfort produced in caring for a larger litter drives the females from their nests more often. A second experiment was, therefore, undertaken in an attempt to test these hypotheses. In this experiment food and water were provided inside the nest so that females did not need to leave the litter in order to obtain nourishment.
391
Methods The subjects of this study were twenty female C3H mice between 14 and 22 weeks of age at the beginning of the experiment. All of the subjects had already successfully reared at least one litter. The mating procedure and housing conditions were identical to those employed in the first experiment and at parturition twelve litters of eight pups and eight litters of two pups were made up using a litter reallocation procedure similar to that used in the earlier study. Due to partial or complete mortality of the litter, one litter of two pups and five litters of eight pups were discarded before the termination of the experiment. Food and water were supplied through the bars that made up the roof of the observation cage and it was possible by manipulating the position of the cage roof to supply food pellets and water directly above the nest, so that females could eat and drink without leaving the litter. The observation procedure was very similar to that employed in the first experiment. Each cage was observed at intervals of 4 min over a period of 4 hr on alternate days of lactation. In this experiment however, the only aspects of behaviour recorded were: (a) whether the female was inside or outside the nest, (b) whether she was nursing, and (c) whether she was eating. Results Scores for the ten observation periods covering the interval between the birth of the litter and the end of the third week of lactation were totalled for each animal, and t-tests were used: (a) to compare the eating, nursing and in-nest scores of both litter size groups when food and water were supplied over the nest with the scores already obtained in the previous study, when food and water were outside in the cage, and (b) to compare the eating, nursing and in-nest scores of females caring for litters of two and eight pups when food was supplied over the nest. The results of these t-tests are summarized in Table II. When food and water were supplied over the nest, females caring for both large and small litters spent significantly more time in the nest than females caring for litters of equivalent size when food and water were outside in the cage (P < 0.01). Females with litters of two pups however, continued to spend significantly more time in the nest than females with litters of eight pups (P < 0-025). Animals rearing litters of both sizes spent slightly less time eating when food was supplied
392
ANIMAL
BEHAVIOUR,
20,
2
Table II. Comparison of Behaviour when Food and Water supplied Inside or Outside the Nest
Litter-size 2 Behaviour No. of observations eating No. of observations Nursing (total) Up to day 14 Days 15-20 No. of observations in nest
Food over nest
Food in cage
Litter-size 8 P
Food over nest
120.3 126.8 0.455
Ns
191.3 224'0
261"3 273.4 0"754*
Ns
213'0
509.4 380.9 8.33*
<0.01
t
Food in cage
t
Food over nest P
2.44 <0'05
248"6 1"38
Ns
431.9 261-6 5-73 <0.01
Litter Litter size size 2 8
t
P
120"3 191.3 5.06 <0.01 261"3 213"0 2"40 <0"05 221"0 162"1 3,96 <0'01 38"9 50'9 0"84 r~s 509.1 431.9 3.63* <0.025
Cells show mean total scores for each experimental group. All probabilities for two-tailed tests. *Heterogeneity of variance-critical value of t calculated by special formula (Edwards 1966. p. 107). overhead than when they had to leave the nest to feed. The difference for females with two pups was significant (P < 0.05), but that for females with eight pups was not. When animals with litters of two and eight pups were compared, it was found that those with larger litters still spent significantly more time eating than those with smaller litters (P < 0.01). The nursing scores of females with litters of two and eight were not significantly different when food was supplied over the nest from the scores when food was outside in the cage, but animals caring for two pups were observed nursing the litter significantly more often than animals caring for eight pups over the first 20 days of lactation (P < 0.05). Discussion
The present studies indicate that litter size has widespread influences upon the behaviour of lactating female mice. While offering some support to the conclusion drawn from earlier investigations with rats, that various aspects of maternal behaviour decrease in incidence as litter size increases (Seitz 1954, 1958; Draper 1968), these studies also suggest that other aspects of behaviour not directly associated with the care of the young are affected. The finding that females caring for smaller litters spend more time in the nest with the pups is consistent with the results of an earlier study with rats (Grota & Ader 1969), though, in the present investigation the differences between experimental groups were apparent immediately after parturition and persisted throughout lac-
tation. The differential nest-attendance of females rearing different sized litters has a number of important implications for the development and later behaviour of the pups. Larger litters may, for example, experience more cooling due to their mother's increased absence from the nest. Early exposure to cold has been shown to have widespread influences upon development (e.g. Barnett & Borland 1967; Gelineo & Gelineo 1952) and there has been some suggestion that temperature change may be the important variable mediating many of the effects on development of additional stimulation in infancy (reviewed by Russell 1971). It is possible, of course, that the cooling of larger litters may be compensated to some extent by the increased heat production and the greater insulation offered by additional littermates, and further studies are required to examine nest temperature and nest temperature fluctuations in different sized litters. It has also been suggested that tactile and kinaesthetic stimulation may be the important variables mediating the effects o f various manipulations in infancy (e.g. Levine 1962). The high nest-attendance of animals rearing smaller litters and the additional licking experienced by the pups in such litters might, therefore, be expected to influence development. Further studies are, at present, being carried out to examine the development and later behaviour of animals reared in litters of different size. Females caring for large litters are known to secrete more milk than females caring for small
PRIESTNALL: LITTER SIZE AND BEHAVIOUR IN THE MOUSE litters, though the quantity of milk available per pup may decrease as litter size increases (Kumaresan, Anderson & Turner 1967). Food consumption appears to parallel milk secretion; Ota & Y o k o y a m a (1967) found a linear relationship between the logarithm of litter size and food intake in lactating female rats. In the present investigation females rearing larger litters were observed eating and drinking on more occasions than females rearing smaller litters and, in a further study by the author (Priestnall 1970), actual food and water consumption was also found to increase with the size of the litter. Though animals rearing larger litters were observed more frequently out of the nest, eating and drinking were the only out-of-nest activities on which they scored higher than animals rearing smaller litters. Resting, grooming, and active, all of which occurred predominantly outside the nest were observed more frequently in females with smaller litters. It seems likely, therefore, that animals with large litters leave the nest more often, and consequently show less maternal behaviour; because of their need for the greater quantities of food and water necessary in order to provide sufficient milk to sustain their large litters. This is not to suggest that other factors are not also involved in accounting for the observed differences in behaviour. Seitz (1958), for example, claimed that fatigue induced in the mothers of larger litters may serve as a 'physiological signal mechanism' activating a stepwise reduction in maternal behaviour as the fatigue level rises. Discomfort produced by excessive contact and interference from the litter, or from the extra stimulation of the m a m m a r y region during suckling could also result in females rearing larger litters leaving the nest more often. An attempt to separate such aversive factors from the need for nourishment was made in the second experiment in which food and water were provided directly above the nest so that the female could eat and drink without leaving her litter. As expected, all animals spent significantly more time in the nest in this study. However, animals rearing larger litters continued to spend more time outside the nest and less time nursing during the first 2 weeks of lactation than animals rearing smaller litters. Unfortunately no data are available on the nature of the behaviour occurring outside the nest but eating and drinking were certainly not involved. It would seem from the second experiment
393
that the differential nest attendance of animals rearing litters o f different sizes cannot be explained simply in terms of the need for food and water; aversive influences may also be operating. It is possible, however, that by forcing the female to eat and drink inside the nest, the second experiment may have magnified the influence of such aversive factors. While the female was eating, she was frequently interfered with by the struggles of the pups and their attempts to initiate nursing. This constant disturbance may have induced more discomfort and fatigue than the comparatively undisturbed feeding situation of the first experiment, thus forcing the mothers of larger litters to rest more frequently outside the nest. Acknowledgments This article is based on a thesis submitted to the Department of Psychology, Leicester University, in partial fulfillment of the requirements of the Ph.D. degree. The author would like to express his gratitude to the Science Research Council, for financing the project, to Dr U. Weidmann for supervising the research, and to Mr G. Evans and Mr D. MacArthur for their technical assistance. R EFERENCES Barnett, S. A. & Borland, K. A. (1967). An effect of cold exposure at birth on the reproduction of mice. J. Reprod. Fert., 13, 501-504. Draper, D. D. (1968). Litter-size, maternal behaviour and pup development in the rat. Ph.D. Thesis, University of Tennessee. Edwards, A. L. (1966). Experimental Design in Psychological Research. New York: Holt, Rinehart & Winston. Gelineo, S. & Gelineo, A. (1952). La temprrature du nid du rat et sa signification biologique. Bull. /cad. serb. Sci. CI. math. nat., 4, 197-210. Grota, L. J. & Ader, R. (1969). Continuous recording of maternal behaviour in Rattus norvegicus. Anim. Behav., 17, 722-729. Hinde, R. A. (1959). Unitary drives. Anita. Behav., 7, 130-141. Hinde, R. A. (1970). Animal Behaviour: A Synthesis of Ethology and Comparative Psychology. New York: McGraw Hill. Kumaresan P., Anderson, R. R. & Turner, C. W. (1967). Effect of litter-size upon milk yield and litter weight gains in rats. Proc. Soc. exp. Biol., 126, 41--45. Levine, S. (1962). The effects of infantile experience on adult behaviour. In: Experimental Foundations of Clinical Psychology (Ed. by A. J. Bachrach). New York: Basic Books. Ota, K. & Yokoyama, A. (1967). Body weight and food consumption of lactating rats nursing various sizes of litter. J. Endocrinol., 38, 263-268.
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Priestnall, R. (1970). Litter size, the mother-infant interaction and adult behaviour in the mouse. Ph.D. Thesis, University of Leicester. Rosenblatt, J. S. & Lehrman, D. S. (1963). Maternal behaviour of the laboratory rat. In: Maternal Behaviour in Mammals (Ed. by H. Reingold). New York: Wiley. Russell, P. A. (1971). 'Infantile stimulation' in rodents: A consideration of possible mechanisms. Psyehol. Bull., '75, 192-202. Seitz, P. F. D. (1954). The effects of infantile experience upon adult behaviour in animal subjects. I. Effects of litter size during infancy upon adult behaviour in the rat. Am. J. Psychiat., 110, 916927.
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Seitz, P. F. D. (1958). The maternal instinct in animal subjects. I. Psychosom. Meal., 20, 215-226. Slotnick, B. M. (1967). Intercorrelations of maternal activities in the rat. Anita. Behav., I5, 267-269. Whitten, W. K. (1958). Modification of the oestrous cycle of the mouse by external stimuli associated with the male. Changes in the oestrous cycle determined by vaginal smears. J. Endocr., 17, 30%313. Whitten, W. K. (1959). Occurrence of anoestrus in mice caged in groups, ar. Endoer., 18, 102-107. Winer, B. J. (1962). Statistical Prineiples in Experimental Design. New York: McGraw Hill. (Received 9 July 1971 ; revised 4 October 1971 ; MS. number: 1077)
EFFECTS OF LITTER SIZE ON THE BEHAVIOUR OF LACTATING FEMALE MICE
(MUS MUSCULUS)
BY ROBIN PRIESTNALL* Department of Psychology, The University, Leicester LE1 7RH Abstract. The behaviour of multiparous female C3H mice rearing different sized litters was examined using an observational time-sampling procedure, The results indicate that while animals rearing smaller litters spend more time in the nest, more time licking individual pups, and during most of lactation, more time nursing the litter, animals rearing larger litters spend more time eating and drinking. It is suggested that the observed differences in behaviour may be the outcome of the differential nutritional requirements arising from feeding litters of different sizes. However, in a second experiment providing food and water inside the nest, the differences in nest-attendance and nursing behaviour persisted, suggesting that some aversive factor such as fatigue or discomfort may also be involved. the time a lactating female rat spends with its litter can be taken as a reflection of maternal behaviour and some support to the validity of this assumption was obtained using a modified version of the maternal behaviour scale devised by Seitz (1958). The present study was designed to investigate both maternal behaviour and behaviour not directly concerned with the care of the young, in mice Mus musculus rearing litters of different sizes. The observational time-sampling technique employed involved no disturbance of either the mother or the offspring, while allowing the separate recording of many aspects of maternal behaviour; a procedure which would seem necessary in view of the low intercorrelations demonstrated between different measures of maternal behaviour (Slotnick 1967) and the general difficulties associated with the unitary drive concept (Hinde 1959, 1970).
The maternal behaviour of rats and, consequently, the early experience of the pups, has been shown to vary with the size of the litter. Seitz (1954, 1958), rating lactating female Wistar rats on nine observational categories, compounded a 'total maternal behaviour score' by addition of the ratings and found that mothers with smaller litters 'behaved significantly more maternally' than mothers with large litters. Seitz's conclusions which are based upon a parametric analysis of the rating-scale data assume implicitly that maternal behaviour is the outcome of a unitary drive. Draper (1968), working with thirteen fitters of primiparous Wistar female rats found that animals caring for smaller litters built more covered nests, while females with larger litters were more likely to move pups away from the nest during tests of retrieving. In this study, females were allowed to rear their own pups and the test procedure, like that of Seitz, involved considerable disturbance of both mothers and offspring. A later investigation by Grota & Ader (1969) employed a dual-chambered nesting cage which recorded electronically the time spent by Charles-River (CD) female rats with the litter in the compartment containing the nest, or away from the litter in the other compartment. The authors report that females caring for both large and small litters spent approximately 85 per cent of their time with the pups immediately after parturition, but the proportion of the time spent with the pups decreased more rapidly over the lactation period in females rearing larger litters. Grota & Ader (1969) assurded that *Present address: Department of Psychology,University of Sheffield,SheffieldSt0 2TN.
Experiment 1 Methods Subjects. The subjects were fifty-eight C3H female mice aged between 16 and 24 weeks at the beginning of the experiment. They had reared their first litters undisturbed and in isolation and were kept in all-female groups of ten to twelve animals for a period of at least 2 weeks before pairing in an attempt to induce oestrous synchrony (Whitten 1958, 1959). Holding conditions. All females were paired simultaneously with single males in small plastic laboratory cages (35 • 15 • 13era), and as soon as it became obvious that they were pregnant (usually during the second week of gestation), the females were transferred to specially constructed observation cages. The 386
PRIESTNALL: LITTER SIZE AND BEHAVIOUR IN THE MOUSE final transfer occurred at least 1 week before parturition and the females were then left undisturbed until the weaning of the litter, except for the manipulation of litter size immediately following parturition. The observation cages were 30 • 25 x 30 cm. The rear and side walls were constructed of 20 gauge galvanized mild steel sheet and the front wall was of Plexiglass. Food was supplied in a 75-mm mesh hopper on the rear wall of the cage and water in a plastic feeder inserted through the Plexiglass observation panel. The floor covering was sawdust and wood shavings, and each cage was supplied with 3 to 4 g of cotton wool for the construction of the nest. Animals were maintained on a diet of 41B animal food pellets. The whole experiment was carried out on a reversed light schedule; the room being in darkness from 09.30 to 19.30 hours. A series of four 100-W red lamps continually burning above the observation cages provided sufficient light for observation during the dark phase. Manipulation of litter size. As parturition became imminent, females were checked at least once every 12 hr and as soon as the litters were born the female and the litter were gently removed from the cage using padded forceps. The litter was placed in a tray of cotton wool heated from below by a 60-W lamp to avoid cooling while the remaining pups were collected. Synchronization of parturition was not totally successful but where two or more litters were present on any check period, the pups were pooled and then both females and pups allocated at random to the three experimental litter-size conditions of two, five and eight pups per litter. Allocation was sometimes limited by the number of pups available. The newly assembled litter was introduced into the nest of its assigned mother and finally, the female was returned to the corner opposite the nest in her own cage. Of the twenty litters of two pups made up in this way, sixteen were reared successfully to weaning. Nine of the fifteen litters of five pups and only three of the .twenty-three litters of eight pups were weaned intact. If one or more pups died during lactation the litter was usually discarded but, because of the high wastage of litters of five and eight pups, some litters suffering only one mortality were retained to increase the sample-size. Three litters of five that were reduced to four pups and four litters of eight reduced to seven were included in this way. A control experiment by the author (Priestnall 1970) revealed that this fostering procedure had
387
no significant influence upon any of the forms of behaviour recorded in this study. Observation of maternal behaviour. Observation was carried out on alternate days over the first 3 weeks of lactation. Females were observed on days 1, 3, 5, 7, etc., or on days 2, 4, 6, 8, etc., depending upon the day of birth of their litters. During the 4-hr observation period, sixty observations were made upon each of the females at intervals of approximately 4 min. This interval allowed sufficient time for observations on up to twenty-five cages to be recorded. During each 4-min cycle the experimenter moved from cage to cage noting on a prepared record sheet the form of behaviour displayed by each female at the moment of observation. The record was made in coded form, the following forms of behaviour being noted: In nest: The female was anywhere inside the nest, indulging in any form of behaviour at the moment of observation. Nursing: The female was allowing the pups to suckle. Nursing did not necessarily imply that the whole litter was nursing, nor that the female was adopting the nursing posture with her body arched over the pups. Licking pups: The female was licking or grooming her pups, Nest building: The female was engaged in some aspect of nest building behaviour at the moment of observation. She could be inside or outside the nest, pushing and kneading material into position with her snout and forepaws, shaping the nest, or pulling nest material towards it. Eating: The female was nibbling at a food pellet. Drinking: The female was drinking from the water container. Grooming: The female was grooming her own body. Resting: The female was lying motionless, either inside or outside the nest, usually with her eyes open but not involved in any other form of behaviour. Active: The female was moving about the cage at the moment of observation. The position of the young (inside or outside the nest) was also recorded together with any form of behaviour not catered for by the code. The next cycle of observation was not commenced until the 4-min interval had elapsed. The whole procedure was then repeated.
388
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Results
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From the record sheets, scores were obtained for each of the classes of behaviour, for each female, on each of the 4-hr observation periods by simply adding the number of observations in which that particular behaviour occurred. Any animal can therefore obtain a score of up to sixty on any class of behaviour, on each day of testing. Scores over the lactation period for each of the forms of behaviour recorded are presented graphically in Figs 1 to 9. The graphs are plotted from the mean scores for each experimental group on alternate days of lactation.
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Data for those classes of behaviour having a high incidence (in nest, nursing, eating, grooming and active) were analysed over the lactation period using an analysis of variance design with repeated measures on one factor. Because of the possibility that the differential mortality observed in different sized litters was related to litter size itself, the least squares solution (Winer 1962) was employed. The results of these analyses are summarized in Table I. For those classes of behaviour having a relatively low incidence (licking pups, licking per pup, nest-building, drinking and resting), total scores were calculated by adding scores over the ten observation periods. The totals were then treated by a single factor analysis of variance and the results of these analyses together with
PRIESTNALL: LITTER. SIZE AND BEHAVIOUR. IN THE MOUSE
389
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the mean total scores and the standard deviations are also available in Table I. Maternal behaviour. Most maternal behaviour occurs inside the nest; at least until the litter becomes active around 2 weeks post-partum. In all litter-size groups nest attendance declined steadily during the first 2 weeks of lactation, showing a slight increase during the third week. This increase was almost certainly due to the progressive flattening and consequent increase in area of the nest, together with the gradual loss of identity of the nest site as distinguished from the
Fig. 8. Resting. rest of the cage. Throughout the entire lactation period females rearing smaller litters were observed more frequently inside the nest. The behaviour most frequently observed in the nest was nursing. Analysis of the nursing scores revealed a highly significant interaction effect. During the first 2 weeks of lactation, females rearing smaller litters obtained the highest nursing scores, but during the third week, females rearing larger litters obtained the higher scores. This interaction effect might be accounted for in terms of a change occurring over the period of lactation in the mother-infant relationship
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increase in m o v e m e n t s by the pups away from the nest, a n d a decrease in the retrieval behaviour of the mother. A t this stage the pups begin to take a more active role i n the initiation of nursing, which they a t t e m p t b o t h inside a n d outside the nest. Pups from larger litters apparently o b t a i n less n o u r i s h m e n t ; they are lighter in weight t h r o u g h o u t the period o f lactation, a n d as a result of limitations o n the female's supply of time a n d milk they are p r o b a b l y rarely satiated. As the onus for initiating n u r s i n g passes to the pups, animals from larger litters may a t t e m p t to do so more frequently t h a n animals from smaller litters, a n d as the pups often solicit n u r s i n g individually outside the nest, they will tend to nurse at different times, with the result that females rearing larger litters will be observed n u r s i n g more frequently at this stage of development. N o significant difference was f o u n d i n the n u m b e r of observations i n which females rearing different sized litters were licking their pups b u t as only one p u p is licked at a time, the p u p ' s experience of licking is better represented by dividing the total licking score by litter size. A n analysis of variance executed after this transf o r m a t i o n indicated that pups reared in smaller litters receive significantly more licking from their mothers.
Table I. Analysis of Behaviour of Females Rearing Litters of Different Size Over the First 3 Weeks of Lactation
Mean total scores Behaviour
Litter size 2
Litter size 5
Litter size 8
Results of analysis of variance Litter size effect
380.9 336'3 261.6 F = 13'69, df = 2/32 (55.4) (40"3) (57.3) P < 0.005 Nursing 273"4 2 9 0 " 6 248.6 F = 20.44, df= 2/32, (51"3) (35"8) (43.9) P < 0.005 Licking pups 13"5 21'7 12-0 F = 2'20, df= 2/32, (4"9) (6"6) (5.3) Ns Licking per pup 6-8 4"3 1.5 F = 19.65, df = 2]32, (1"9) (1"4) (0'2) P = 0.005 Nest-building 9"8 20"3 16-4 F = 4"82, df = 2/32, (8-0) (7"6) (7.9) P < 0.05 Eating 126.8 142"0 224.0 F = 30.53, df = 2/32, (31'6) (20"6) (29.7) P < 0.005 Drinking 8.0 11"3 43.0 F = 11-46, df= 2/32, (3.5) (3"5) (32-3) P < 0.005 Grooming 96"2 82'8 69.0 F = 7.40, df = 2/32, (17.7) (19"8) (14.5) P < 0"005 Resting 54.4 18"3 10.7 F = 20.12, df= 2/32, (24.6) (9.8) (9-5) P < 0.005 Active 48.4 42'3 31-6 F ~ 1'39, df ~ 2/32, (28.4) (13"2) (17.7) NS The numbers in parentheses are standard deviations.
In nest
Days e f f e c t
Interaction effect
F = 29.85, df = 9/288 F = 1.36, df = 18/288 P < 0.005 Ns F = 49.28, df= 9/288, F = 496.51, df= 181288, P < 0.005 P < 0.005 ----
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F = 37.03, df = 9/288, F = 2.70, df= 181288, P < 0.005 P < 0.005 --F = 2.41, df = 9/288, P < 0.05 --
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F = 3.62, df = 9/288, P < 0.005
F -- 1.93, df= 181288, P < 0"05
PRIESTNALL: LITTER SIZE AND BEHAVIOUR IN THE MOUSE The statistical analysis suggests that females caring for the intermediate sized litters of five pups spend the most time involved in building and repairing the nest ( F = 4-82, df= 2/32, P < 0.05). However, the relatively low incidence of this form of behaviour and the high standard deviations of the mean total scores reduce the reliability of this finding. Nest building declined steadily in incidence over lactation, disappearing almost completely in all groups by the end of the second week post-partum. Non-maternal behaviour. The most frequently observed form of behaviour outside the nest was eating. Eating appeared to reach peak incidence in females in the large (litter size 8) and small (litter size 2) groups about 14 days after the birth of the litter. In the intermediate (litter size 5) group peak incidence occurred a little later, probably accounting for the significant interaction effect on this measure. The decline in food intake during the third week of lactation is probably related to changes in the rate of growth of the pups and to the transition of the litter onto solid food at this stage. Throughout lactation animals rearing larger litters were observed eating more frequently than animals rearing smaller litters, and drinking behaviour though much lower in incidence, showed a similar pattern; animals with larger litters being observed drinking significantly more often. On the other forms of non-maternal behaviour recorded; grooming, resting, and activity, females rearing smaller litters obtained higher scores, the differences in the grooming and resting scores being significant. A significant interaction effect was obtained on the activity score ( F - - 1.93, df= 18/288, P < 0.05) but in view of the low incidence of this class of behaviour and the great variability in the scores, this finding may not be reliable.
Experiment 2 The first experiment suggests that females caring for larger litters leave the nest more often, and consequently show less maternal behaviour, because of their greater need for food and water. Another possibility is that some aversive factor such as fatigue or discomfort produced in caring for a larger litter drives the females from their nests more often. A second experiment was, therefore, undertaken in an attempt to test these hypotheses. In this experiment food and water were provided inside the nest so that females did not need to leave the litter in order to obtain nourishment.
391
Methods The subjects of this study were twenty female C3H mice between 14 and 22 weeks of age at the beginning of the experiment. All of the subjects had already successfully reared at least one litter. The mating procedure and housing conditions were identical to those employed in the first experiment and at parturition twelve litters of eight pups and eight litters of two pups were made up using a litter reallocation procedure similar to that used in the earlier study. Due to partial or complete mortality of the litter, one litter of two pups and five litters of eight pups were discarded before the termination of the experiment. Food and water were supplied through the bars that made up the roof of the observation cage and it was possible by manipulating the position of the cage roof to supply food pellets and water directly above the nest, so that females could eat and drink without leaving the litter. The observation procedure was very similar to that employed in the first experiment. Each cage was observed at intervals of 4 min over a period of 4 hr on alternate days of lactation. In this experiment however, the only aspects of behaviour recorded were: (a) whether the female was inside or outside the nest, (b) whether she was nursing, and (c) whether she was eating. Results Scores for the ten observation periods covering the interval between the birth of the litter and the end of the third week of lactation were totalled for each animal, and t-tests were used: (a) to compare the eating, nursing and in-nest scores of both litter size groups when food and water were supplied over the nest with the scores already obtained in the previous study, when food and water were outside in the cage, and (b) to compare the eating, nursing and in-nest scores of females caring for litters of two and eight pups when food was supplied over the nest. The results of these t-tests are summarized in Table II. When food and water were supplied over the nest, females caring for both large and small litters spent significantly more time in the nest than females caring for litters of equivalent size when food and water were outside in the cage (P < 0.01). Females with litters of two pups however, continued to spend significantly more time in the nest than females with litters of eight pups (P < 0-025). Animals rearing litters of both sizes spent slightly less time eating when food was supplied
392
ANIMAL
BEHAVIOUR,
20,
2
Table II. Comparison of Behaviour when Food and Water supplied Inside or Outside the Nest
Litter-size 2 Behaviour No. of observations eating No. of observations Nursing (total) Up to day 14 Days 15-20 No. of observations in nest
Food over nest
Food in cage
Litter-size 8 P
Food over nest
120.3 126.8 0.455
Ns
191.3 224'0
261"3 273.4 0"754*
Ns
213'0
509.4 380.9 8.33*
<0.01
t
Food in cage
t
Food over nest P
2.44 <0'05
248"6 1"38
Ns
431.9 261-6 5-73 <0.01
Litter Litter size size 2 8
t
P
120"3 191.3 5.06 <0.01 261"3 213"0 2"40 <0"05 221"0 162"1 3,96 <0'01 38"9 50'9 0"84 r~s 509.1 431.9 3.63* <0.025
Cells show mean total scores for each experimental group. All probabilities for two-tailed tests. *Heterogeneity of variance-critical value of t calculated by special formula (Edwards 1966. p. 107). overhead than when they had to leave the nest to feed. The difference for females with two pups was significant (P < 0.05), but that for females with eight pups was not. When animals with litters of two and eight pups were compared, it was found that those with larger litters still spent significantly more time eating than those with smaller litters (P < 0.01). The nursing scores of females with litters of two and eight were not significantly different when food was supplied over the nest from the scores when food was outside in the cage, but animals caring for two pups were observed nursing the litter significantly more often than animals caring for eight pups over the first 20 days of lactation (P < 0.05). Discussion
The present studies indicate that litter size has widespread influences upon the behaviour of lactating female mice. While offering some support to the conclusion drawn from earlier investigations with rats, that various aspects of maternal behaviour decrease in incidence as litter size increases (Seitz 1954, 1958; Draper 1968), these studies also suggest that other aspects of behaviour not directly associated with the care of the young are affected. The finding that females caring for smaller litters spend more time in the nest with the pups is consistent with the results of an earlier study with rats (Grota & Ader 1969), though, in the present investigation the differences between experimental groups were apparent immediately after parturition and persisted throughout lac-
tation. The differential nest-attendance of females rearing different sized litters has a number of important implications for the development and later behaviour of the pups. Larger litters may, for example, experience more cooling due to their mother's increased absence from the nest. Early exposure to cold has been shown to have widespread influences upon development (e.g. Barnett & Borland 1967; Gelineo & Gelineo 1952) and there has been some suggestion that temperature change may be the important variable mediating many of the effects on development of additional stimulation in infancy (reviewed by Russell 1971). It is possible, of course, that the cooling of larger litters may be compensated to some extent by the increased heat production and the greater insulation offered by additional littermates, and further studies are required to examine nest temperature and nest temperature fluctuations in different sized litters. It has also been suggested that tactile and kinaesthetic stimulation may be the important variables mediating the effects o f various manipulations in infancy (e.g. Levine 1962). The high nest-attendance of animals rearing smaller litters and the additional licking experienced by the pups in such litters might, therefore, be expected to influence development. Further studies are, at present, being carried out to examine the development and later behaviour of animals reared in litters of different size. Females caring for large litters are known to secrete more milk than females caring for small
PRIESTNALL: LITTER SIZE AND BEHAVIOUR IN THE MOUSE litters, though the quantity of milk available per pup may decrease as litter size increases (Kumaresan, Anderson & Turner 1967). Food consumption appears to parallel milk secretion; Ota & Y o k o y a m a (1967) found a linear relationship between the logarithm of litter size and food intake in lactating female rats. In the present investigation females rearing larger litters were observed eating and drinking on more occasions than females rearing smaller litters and, in a further study by the author (Priestnall 1970), actual food and water consumption was also found to increase with the size of the litter. Though animals rearing larger litters were observed more frequently out of the nest, eating and drinking were the only out-of-nest activities on which they scored higher than animals rearing smaller litters. Resting, grooming, and active, all of which occurred predominantly outside the nest were observed more frequently in females with smaller litters. It seems likely, therefore, that animals with large litters leave the nest more often, and consequently show less maternal behaviour; because of their need for the greater quantities of food and water necessary in order to provide sufficient milk to sustain their large litters. This is not to suggest that other factors are not also involved in accounting for the observed differences in behaviour. Seitz (1958), for example, claimed that fatigue induced in the mothers of larger litters may serve as a 'physiological signal mechanism' activating a stepwise reduction in maternal behaviour as the fatigue level rises. Discomfort produced by excessive contact and interference from the litter, or from the extra stimulation of the m a m m a r y region during suckling could also result in females rearing larger litters leaving the nest more often. An attempt to separate such aversive factors from the need for nourishment was made in the second experiment in which food and water were provided directly above the nest so that the female could eat and drink without leaving her litter. As expected, all animals spent significantly more time in the nest in this study. However, animals rearing larger litters continued to spend more time outside the nest and less time nursing during the first 2 weeks of lactation than animals rearing smaller litters. Unfortunately no data are available on the nature of the behaviour occurring outside the nest but eating and drinking were certainly not involved. It would seem from the second experiment
393
that the differential nest attendance of animals rearing litters o f different sizes cannot be explained simply in terms of the need for food and water; aversive influences may also be operating. It is possible, however, that by forcing the female to eat and drink inside the nest, the second experiment may have magnified the influence of such aversive factors. While the female was eating, she was frequently interfered with by the struggles of the pups and their attempts to initiate nursing. This constant disturbance may have induced more discomfort and fatigue than the comparatively undisturbed feeding situation of the first experiment, thus forcing the mothers of larger litters to rest more frequently outside the nest. Acknowledgments This article is based on a thesis submitted to the Department of Psychology, Leicester University, in partial fulfillment of the requirements of the Ph.D. degree. The author would like to express his gratitude to the Science Research Council, for financing the project, to Dr U. Weidmann for supervising the research, and to Mr G. Evans and Mr D. MacArthur for their technical assistance. R EFERENCES Barnett, S. A. & Borland, K. A. (1967). An effect of cold exposure at birth on the reproduction of mice. J. Reprod. Fert., 13, 501-504. Draper, D. D. (1968). Litter-size, maternal behaviour and pup development in the rat. Ph.D. Thesis, University of Tennessee. Edwards, A. L. (1966). Experimental Design in Psychological Research. New York: Holt, Rinehart & Winston. Gelineo, S. & Gelineo, A. (1952). La temprrature du nid du rat et sa signification biologique. Bull. /cad. serb. Sci. CI. math. nat., 4, 197-210. Grota, L. J. & Ader, R. (1969). Continuous recording of maternal behaviour in Rattus norvegicus. Anim. Behav., 17, 722-729. Hinde, R. A. (1959). Unitary drives. Anita. Behav., 7, 130-141. Hinde, R. A. (1970). Animal Behaviour: A Synthesis of Ethology and Comparative Psychology. New York: McGraw Hill. Kumaresan P., Anderson, R. R. & Turner, C. W. (1967). Effect of litter-size upon milk yield and litter weight gains in rats. Proc. Soc. exp. Biol., 126, 41--45. Levine, S. (1962). The effects of infantile experience on adult behaviour. In: Experimental Foundations of Clinical Psychology (Ed. by A. J. Bachrach). New York: Basic Books. Ota, K. & Yokoyama, A. (1967). Body weight and food consumption of lactating rats nursing various sizes of litter. J. Endocrinol., 38, 263-268.
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Priestnall, R. (1970). Litter size, the mother-infant interaction and adult behaviour in the mouse. Ph.D. Thesis, University of Leicester. Rosenblatt, J. S. & Lehrman, D. S. (1963). Maternal behaviour of the laboratory rat. In: Maternal Behaviour in Mammals (Ed. by H. Reingold). New York: Wiley. Russell, P. A. (1971). 'Infantile stimulation' in rodents: A consideration of possible mechanisms. Psyehol. Bull., '75, 192-202. Seitz, P. F. D. (1954). The effects of infantile experience upon adult behaviour in animal subjects. I. Effects of litter size during infancy upon adult behaviour in the rat. Am. J. Psychiat., 110, 916927.
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Seitz, P. F. D. (1958). The maternal instinct in animal subjects. I. Psychosom. Meal., 20, 215-226. Slotnick, B. M. (1967). Intercorrelations of maternal activities in the rat. Anita. Behav., I5, 267-269. Whitten, W. K. (1958). Modification of the oestrous cycle of the mouse by external stimuli associated with the male. Changes in the oestrous cycle determined by vaginal smears. J. Endocr., 17, 30%313. Whitten, W. K. (1959). Occurrence of anoestrus in mice caged in groups, ar. Endoer., 18, 102-107. Winer, B. J. (1962). Statistical Prineiples in Experimental Design. New York: McGraw Hill. (Received 9 July 1971 ; revised 4 October 1971 ; MS. number: 1077)