Alterations in the Maternal Behavior of Rats Rearing Malnourished Offspring SANDRA G. WIENER KATHLEEN M. FITZPATRICK ROBERT LEVIN WILLIAM P. SMOTHERMAN SEYMOUR LEVINE Department of Psychiatry and Behavioral Sciences Stanford University School o f Medicine Stanford, California Investigations of maternal behavior of mothers fed a low-protein diet indicated deficits in retrieval and in the rate of nest-building. In addition, they indicated a concomitant increase in time spent with young when assessed during periods not associated with the retrievaI/nest-building test session. The adrenalectomized mother, another case that produces growth-stunted progeny, was compared with both low-protein and control mothers for maternal behavior. Unlike the low-protein mother, the adrenalectomized mother did not exhibit retrieval or nest-building deficits; however, the adrenalectomized mother did display an increase in time spent with young. These data suggest that although deficits in retrieval and nest-building can be attributed to the nutritional condition of the mother, the stimulus characteristics of the malnourished pup are important in eliciting the increased time spent with the litter.
Recently, a number of reviews of the effects of perinatal malnutrition on later behavior in rodents have pointed out that the laboratory methods used to produce early malnutrition are confounded by variations in other environmental factors, such as amount of early handling or disruption of the mother-infant relationship (Levine & Wiener, 1975; Winick & Coombs, 1972). Because changes in these environmental factors, independent of any change in the nutritional state of the neonate, can produce long-lasting behavioral differences in adulthood (Daly, 1973 ; Denenberg, 1969 ; Newton & Levine, 1968), these nonnutritional variables must be carefully controlled so that effects of perinatal malnutrition can be properly evaluated. Several of the methods commonly used to produce malnutrition in rodents (e.g., restricting the time pups spend with the mother, increasing the litter size to 15-20 pups) produce obvious disruptions in the mother-infant relationship (see reviews by Levine & Wiener, 1975; Plaut, 1970). Another technique, restricting the total food intake of the lactating mother by feeding 50% of normal intake once a day, produces deficits in maternal retrieval behavior and shifts the normal circadian rhythm of maternal care (Smart & Preece, 1973; Stern & Levin, 1976).
Reprint requests should be sent to Dr. Sandra G. Wiener, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305. Received for publication: 19 April 1976 Revised for publication: 14 June 1976 Developmental Psychobiology, 10(3): 243-254 (1 977) @ 1977 by John Wiley & Sons, Inc.
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Recent investigations of the maternal behavior of mothers fed a low-protein diet ad libitum (Frahkova, 1971, 1974; Massaro, Levitsky, & Barnes, 1974) have demonstrated both qualitative and quantitative changes in maternal behavior. Fraiikova reported that during 3-min tests on Days 6, 8, and 10 postpartum low-protein mothers retrieve fewer pups with a prolonged latency t o retrieve the first pup. Further, Frahkova observed that low-protein mothers spend less time with their litter and explore more during retrieval tests. On the basis of these observed decreases in 3, puporientcd behaviors, she concluded that low-protein mothers display a deficit in maternal attention toward their litters. On the other hand, Massaro e t al. reported that when undisturbed low-protein mothers are observed by photographic means during the 12-hr dark period, they spend more time with their young than similarly observed control dams. These investigators attributed the inconsistency of these latter observations with those reported by Frafikova to discrepancies in methodology. Together, these studies raise the questions whether low-protein mothers tested for retrieval display an increased amount of time with their young during periods not associated with the retrieval test period and whether the daily test procedures result in an overall decrease in maternal attention toward the litter. The following studies were undertaken t o characterize the low-protein mother’s retrieval behavior and nest-building ability, and to assess the time spent with young during periods not associated with the retrieval/nest-building tests. Whether alterations in the low-protein dam’s maternal behavior are due to her own malnourished condition or her raising malnourished pups was also investigated.
Experiment 1 The purpose of this experiment was t o assess retrieval, nest-building, and contact with offspring during periods not associated with the retrieval test for mothers fed a low-protein diet and to compare these behaviors with those of mothers fed a control diet. Additional sets of mothers not tested for retrieval or nest-building ability were observed for contact with young to control for any disruption of normal mother-infant relationship resulting from these tests.
Subjects Female Sprague-Dawley rats (Rattus nowegicus) received from Simonsen Laboratories (Gilroy, California) at approximately 75 days o f age were housed in groups of 3 in metal pans (28 x 48 x 1 5 cm). Seven days after their arrival, they were mated with Long-Evans males (Simonsen Laboratories, Gilroy, California) t o obtain a genetic hybrid offspring. Daily vaginal smears were taken and the presence of sperm marked the 1st day, Day 0, of gestation. On the 14th day of gestation, females were individually housed in clear plastic cages (28 x 48 x 24 cm) that had a flooring of wood shavings and a grid top that held food pellets and a water bottle. Whereas half of the females (n = 16) continued to be maintained on the stock laboratory diet until weaning (23% protein diet; Feedstuffs Processing Co., San Francisco, California), the other half (n = 16) were placed on a low-protein, isocaloric diet (85%protein diet; Teklad Mills, Madison, Wisconsin),
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on which they were maintained until weaning. On the 20th day of gestation each female was given a packet of newspaper strips for nesting material. Females were checked twice daily for the presence of pups and within 12 hr of parturition (Postpartum Day 0) litters were adjusted to 8 pups (4 males and 4 females). At weaning (Postpartum Day 21), all litters were weighed. Mothers which failed to rear at least 6 pups until weaning were not included in the study. All subjects were kept in an air-conditioned, constant temperature and humidity colony room with controlled lights on a 12-hr on/l2-hr off cycle. Food and water were continuously available. Cages were cleaned at parturition but were subsequently left uncleaned until weaning. Mothers within each nutrition group were assigned alternately to either a group tested for retrieval behavior or a nontested group. This resulted in 4 groups (n = 8 per group): dams fed a low-protein diet and tested for retrieval behavior (LP-T); dams fed a low-protein diet and not tested for retrieval behavior (LP-NT); dams fed a control diet and tested for retrieval (C-T); and dams fed a control diet and not tested for retrieval (C-NT).
Met hods Each day mothers in the LP-T and C-T groups were tested for retrieval behavior at 1% hr before lights-on. For these tests the maternity cages were quietly moved from the colony room to an adjacent anteroom. Both rooms were illuminated by dim red light that remained on only during the testing period t o facilitate observations. In the anteroom dams were placed in holding pans for 2-3 min while their young were removed from the nest and placed in a plastic holding container. The young were then scattered over the floor of the maternity cage opposite the nest. Mothers were then returned to their nest and retrieval of each pup was noted. These observations were continued until the entire litter was retrieved or until 10 min had elapsed. At the end of the retrieval test, the nesting material (paper strips) was removed from the maternity cages and replaced with a fresh packet of nesting material. Cages were then returned to the colony room. At 1 hr and 5 hr after placement of fresh nesting material, cages were examined for the presence of a nest. An animal was scored as having a nest if the paper strips were piled in a circular or semicircular mound to a height of at least 4-5 cm. Maternal contact with young was assessed twice daily in both retrieval tested and nontested groups. These assessments were made once during the dark period under dim red illumination (4 hr before lights-on) and once during the light period (4 hr after lights-on). Each testing period was 30 min long with 15 observations (1 every 2 min) of each mother’s behavior. During each check a female was scored as being in contact with her litter if she was in the nest area with at least 4 pups. Observations of retrieval, nest-building, and maternal contact with young were carried out on Days 2 through 12 postpartum. The decline in the observed maternal retrieval and nest-building behavior and the concomitant increase in offspring activity prevented reliable observations after the 2nd week postpartum. Although the untransformed data are presented in the figures, the frequency of occurrence for the various categories of maternal behavior were subjected to arcsin
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transformations before analysis. The transformed scores for retrieval were analyzed by a 2 (Maternal Nutrition) x 1 1 (Days Postpartum) analysis of variance with the Days factor treated as a within subjects factor. The nest-building data were subjected t o a 2 (Maternal Nutrition) x 11 (Days) x 2 (Time of Observation) analysis of variance with the last 2 factors treated as within subjects factors. Maternal contact was analyzed by a 2 (Maternal Nutrition) x 2 (Tested/Nontested) x 11 (Days) x 2 (Time of Observation) analysis of variance with the last 2 factors treated as within subjects factors.
Results and Discussion The offspring of LP mothers weighed significantly less at weaning than the young of C mothers ( F =532.02, df = 1/288, p < ,001). These body weights (LP offspring: 17.38 2 1.00 g; C offspring: 46.88 ? .84 g) are similar t o those reported for pups raised b p mothers fed a low-protein diet or control diet during lactation (Massaro et al., 1974; Shoemaker & Wurtman, 1971). Summed observations over days showed that LP mothers retrieved fewer pups during a 10-niin retrieval test when compared t o C mothers ( F = 4.37, df = 1/14, I J < .05; Fig. 1). However, the significant Maternal Nutrition x Days interaction (I; = 2.34, df = 10/140, p < .02) indicated that although the retrieval behavior of LP mothers did not differ from C mothers during the early days postpartum, these LP mothers retrieved a significantly smaller percentage of their litter ( p < . O l ) beginning on Day 9 postpartum (post hoc tests for simple main effects; Winer, 1971, p. 347). Thus, LP mothers showed a deficit in this measure of retrieval behavior which became apparent only after the first week postpartum.
Fig 1 . Daily percentage of litter retrieved by control (C) and low-protein (LP) mothers during a l0-niin retrieval test on Days 2-1 2 postpartum (C > LP on Days 9-1 2).
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Fig. 2. Percentage of control (C) and low-protein (LP) mothers with nests within 1 hr and 5 hr of receiving nestbuilding material (X f S.E.).
A significant Maternal Nutrition x Time of Observation interaction ( F = 24.08, d f = 1/28, p < .001) was found in the nest-building behavior of LP and C mothers (see Fig. 2). Further analysis of this effect demonstrated that LP mothers built fewer nests than controls 1 hr after being presented with a new packet of nesting material ( p < .OS) but did not differ from controls at 5 hr. Thus, the disruption in nest-building behavior appeared to be transient reflecting, perhaps, the rate not the ability of low-protein mothers to construct a nest. Analyses of the mothers' behavior during the 2 daily observation periods yielded a number of significant effects. As previously reported by Grota and Ader (1969), mothers spend more time with their litters during the lights-on period and the significant Time of Observation effect bears this out ( F = 128.95, df = 1/28, p < .001). However, because Time of Observation did not interact with Maternal Nutrition or Days, only the average daily score is reported here. The main effect of Maternal Nutrition ( F = 65.06, df = 1/28, p < .OOl) and the significant Maternal Nutrition x Days interaction ( F = 3.30, df = 10/28. p < .Ol) indicate that the LP mothers spent more time in contact with their offspring on postpartum Days 3-12 (see Fig. 3). This was the case whether or not the mother was subjected to a daily retrieval/nest-building test ( F = 1.64, d f = 1/28, p > .05). Whereas the C mothers showed a characteristic decrease in time spent with young as the pups grew older (Rosenblatt, 1965), the LP mothers maintained a higher level of maternal contact during these days.
Experiment 2 Although Experiment 1 demonstrated alterations in the maternal behavior of low-protein mothers, it did not indicate whether these alterations were due t o the
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mother’s malnourished condition or the size and stimulus characteristics of the pups she reared. Rodent offspring are n o t passive recipients of tnaternal care. but provide cues that organize and coordinate mother-infant behavior sequences (Noirot, 1972; Reisbick, Rosenblatt, & Mayer, 1975; Smotherman, Bell, Starzec, Elias, & Zachman, 1974). Possibly, the increased time low-protein mothers spend with young may be elicited by the small size of her pups, which maintain an earlier postpartum form of maternal behavior characterized by more maternal contact (Rosenblatt, 1965). However, if the stimulus characteristics of the growth-stunted pups are the only factor, we would expect that these characteristics would also elicit an adequate pattern of retrieval and nest-building. Obviously, this is not the case because the low-protein mothers display a deficit in these 2 behaviors. Thus. the mother’s nutritional state must also play an important role in determining the alterations in these maternal behaviors. The purpose of Experiment 2 was t o compare the maternal behavior of lowprotein mothers t o the behavior of another type of mother that also raises growthstunted pups: an adrenalectoniized mother. Adrenalectomy was chosen as a preparation because of previous work demonstrating that adrenalectomized mothers rear offspring of similar body weight t o those reared b y low-protein mothers (Thoman, Sproul, Seeler. & Levine, 1970). Although the absence of adrenal glucocorticoids is resporisible for the reduction in lactation (Cowie & Folley, 1947), the presence of a functioning adrenal is not necessary for the initiation or maintenance of maternal responsiveness in rats (Thoman & Levine, 1970). Adrenalectomized mothers deliver young, show maternal behavior, and rear viable offspring.
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Subjects The subjects of this experiment were Sprague-Dawley female rats obtained, housed, mated, and rehoused in the same manner as in Experiment 1. In addition to the low-protein (LP) and control (C) diet mothers, a set of mothers that were adrenalectornized (ADX) were studied. On Day 16-17 of gestation, mothers assigned t o the ADX group underwent surgery under ethyl-ether anesthesia. The animal's back was shaved, and a 4-cm dorsal incision was made. The peritoneum on the right side was then punctured at the posterior end of the rib cage, and a retractor was inserted to permit exposure of the adrenal gland. The adrenal was excised with curved forceps and examined t o assure that it had not been ruptured. If damage to the adrenal had occurred, the animal was discarded. This procedure was then carried out on the animal's left side, after which the peritoneum openings were closed with 5-0 silk suture. Wound clips were used t o close the mother's back and penicillin (Bicillin, Wyeth) was administered. Salt licks were provided to compensate for the lack of salt regulation resulting from adrenalectomy. Completeness of adrenalectomy was determined by plasma corticosteroid levels after ether stress on Day 14 postpartum and females showing significant steroid elevations were not included in the analyses of maternal behavior. At parturition, mothers within LP, C, and ADX groups were divided into groups tested for retrieval behavior (LP-T, C-T, ADX-T [n = 7 per group]) or nontested groups (LP-NT, C-NT, ADX-NT [n = 7 per group] ).
Methods The procedures used to test retrieval, nest-building, and maternal contact with young were identical to those used in Experiment 1. Body weights of the pups from LP-T, C-T, and ADX-T mothers were recorded daily just prior t o the retrieval testing. These weights were analyzed by a 2-factor, 3 (Offspring Type) x 11 (Days of Age), analysis of variance with the Days factor treated as a within subjects factor. The maternal behavior data were analyzed as described in Experiment 1, with the addition of the 3rd maternal group (ADX). Tests on simple main effects and subsequent comparisons by Newman-Keuls procedures were carried out where appropriate.
Results and Discussion The main effect of Offspring Type ( F = 73.86, df = 2/18, p < .OOl) and the interaction of Offspring Type x Age ( F = 72.71, df = 20/180, p < .001) reflect the different growth rates for each Offspring Type (see Fig. 4). Tests for simple effects of Offspring Type a t each age yielded the following results: progeny of LP and ADX mothers weighed less than controls on Days 3-12 (p < .Ol), whereas the offspring of ADX mothers did not begin to differ from LP young until Day 9 (p < .05). Thus, the LP and ADX mothers raised pups of similar size (within 2-3 g) throughout the entire testing period.
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The main effect of Maternal Group (F = 5.30, d f = 2/18, p < .02) and the Materd f = 20/180, p < ,001) indicate, as in Experiment 1 , that the retrieval performance of LP mothers differed froin that of the controls (see Fig. 5). In this experiment, however, differences were already present on Day 6 postpartum ( p < .0S), On the other hand, ADX mothers did not demonstrate the decline in retrieval behavior characteristic of the LP mothers. The maintenance of normal retrieval behavior in ADX mothers confirms and extends the previous report by Thoman and Levine (1970). The nest-building behavior of ADX mothers was indistinguishable froin controls at both the 1-hr and 5-hr intervals (see Fig. 6). Again the LP mothers showed deficits in the number of nests constructed within 1 hr after presentation of nesting materials, which resulted in the significant Maternal Group x Timc of Observation interaction ( F = 35.85, d f = 2/36, p < ,001). Thus, the disruption of retrieval and nest-building behavior in the LP mothers appears not to be due only t o the size of her offspring, but may be a direct effect of the mother’s nutrition on her maternal behavior. The ADX mothers, raising similar size pups, did not display retrieval or nest -building deficits. The analysis of maternal contact with young indicated a main effect of Maternal Group ( F = 45.42, d f = 2 / 3 6 , p < .001); however, the Maternal Group x Days interaction iial Group x Days interaction ( F = 2.89,
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Fig. 5. Daily percentage of litter retrieved by control (C), low-protein (LP), and adrenalectomized (ADX) mothers during a 10-min retrieval test on Days 2-12 postpartum (C > LP on Days 6-12; ADX > LP on Days 8-12).
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Fig. 6 . Percentage of control (C), low-protein (LP), and adrenalectomized (ADX) mothers with i S.E.). nests within 1 hr and 5 hr of receiving nest-building material
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Fig. 7 . Daily mean percent of time control (C), low-protein (LP), and adrenalectonlized (ADX) mothers were observed in contact with litter during two 30-min observation periods o n Days 2-12 postpartum (C < LP, ADX on Days 4-1 2).
was also significant ( F = 3.31, d f = 20/360, p < .01; Fig. 7). In contrast with the results for retrieval and nest-building, the ADX mothers resembled LP mothers on the measure of contact with young. Both LP and ADX mothers showed an increase in the amount of time spent with young when compared t o C mothers on Days 4-12 postpartum O-, < .05). Thus, unlike retrieval and nest-building, contact with young appears t o be related to the stimulus qualities of the pups (e.g., their small size, suckling characteristics, ultrasonic vocalizations, or lack of mobility due to retarded neuromotor development).
General Discussion In this report we have demonstrated that mothers fed a low-protein diet exhibit deficits in retrieval during the 2nd week of lactation, a deficit in their rate of building a nest, and an increase in time spent with young. These results not only confirm the results of Frahkovi (1971, 1974) and Massaro et al. (1974), but also demonstrate that these alterations in various measures of maternal behavior occurred concomitantly. The low-protein mother may under certain circumstances (e.g., during a retrieval test) display decreased attention toward her offspring; however, this does not generalize t o an overall decrease in time spent with her pups during those periods when she is relatively undisturbed by the experimenter. The comparison of the maternal behavior of the low-protein and adrenalectoniized dams suggests that both the mother's nutritional status and her pup's malnutrition play a role in determining the material behavior of the low-protein mother. On the
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one hand, although the adrenalectomized dams raised pups of similar size, they did not display the deficits in retrieval or nest-building of low-protein mothers. This finding suggests that the malnourished condition of the low-protein mother is at least partly responsible for these deficits. Whether malnutrition directly affects retrieval and nest-building or whether these alterations reflect a more general susceptibility to disturbance in their environment remains to be determined. Support for the latter is provided by the appearance of retrieval deficits only after repeated testing (during the 2nd week of lactation). These experiments leave the question unanswered whether the repeated testing and the disturbance accompanying the daily retrieval and nestbuilding test procedure are responsible for the decline in retrieval or whether the decline occurs on Days 6-8 regardless of past testing experience. On the other hand, the stimulus characteristics of the pups appear to be the important factor in eliciting an increase in maternal contact in that both adrenalectomized and low-protein mothers displayed this alteration in maternal behavior. A recent report (Lynch, 1976) on a nipple-ligation method to produce malnourished offspring has indicated that this type of mother raising growth-stunted young spends an increased amount of time with her litter. Thus, any preparation used to produce perinatal malnutrition in rodent offspring will probably induce mothers to spend increased time with young unless some other behavior competes for the mother’s attention. This would probably account for the decreased time spent with young observed in the intake-restricted mother (Smart & Preece, 1973). These restricted mothers spent less time with their offspring compared t o nonrestricted mothers during observations preceding the daily food presentation, but spent more time with the litter during the succeeding periods. The importance of these alterations in the low-protein dam’s maternal behavior in relation to the later behavioral development of the offspring is yet to be determined. However, if the alterations we have shown in the mother’s behavior result in a disruption of the mother-infant relationship, then any long-term influence on the pup’s development attributed directly to early malnutrition must be viewed with caution. These effects could as easily be attributed to other demonstrable influences of early experience on later behavior.
Notes This research was supported by Biosciences Training Grant MH-8304 from t h e National Institute of Mental Health (to S. G. Wiener, R. Levin, and W. P. Smotherman), Research Grant NICH&HD-02881 from the National Institutes of Health, and USPHS Research Scientist Award K5MH-19936 from the National Institute of Mental Health (S. Levine). S. Wiener is now at The Salk Institute, San Diego, CA.
References Cowie, A. T., and Folley, S. I. (1947). Adrenalectomy and replacement therapy in lactating rats. 2 . Effects of deoxycorticosterone acetate o n lactation in adrenalectomized rats. J. Endocrind, 5: 14-23. Daly, M. (1973). Early stimulation of rodents: A critical review of present interpretations. Brit. J. P S J J C ~ 64: O ~ .435-460. ,
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Denenberg, V. H. (1969). The effects of early experience. In E. S. E. Hafez (Ed.), The Behaviour of Domestic Animals. London: Bailliere, T i n d a l l k Cassell. Pp. 109-138. I:rafikovi, S. (1971). Relationship between nutrition during lactation and maternal behaviour of rats. Activ. nerv. sup., 13: 1-8. Frahkovi, S. (1974). Effects of protein deficiency in early life and during lactation on maternal behaviour. Baroda J. Nutr.. I : 21-28. Grata, L. J., and Ader, R. (1969). Continuous recording of maternal behaviour in Kattus norvegfcus. Anim. Behav., 1 7 : 722-729. Levine, S., and Wiener, S. (1975). A critical analysis of data o n malnutrition and behavioral deficits. Adv. Pediat., 22: 11 3-1 36. Lynch, A. (1976). Postnatal undernutrition: An alternative method. Del,. Psychohiol., 9 : 39-48. Massaro, T . F., Levitsky, D. A,, and Barnes, A. H. (1974). Protein malnutrition in t h e rat: Its et'fects on maternal behavior and pup development. Dev. Psychohiol., 7: 551-561. Newton, G., and Levine, S. (1968). Early Experience and Behavior. Springfield, Ill.: C. C. Thomas. Noirot, E. (1972). The onset of maternal behavior in rats, hamsters, and mice. A selective review. Adv. Study Behav., 4 : 107-145. Plaut, S. M. (1970). Studies of undernutrition in the young rat: Methodologiwl considerations. Dev. Psychobiol., 3 : 157-167. Keisbick, S., Rosenblatt, J. S., and Mayer, A . D. (1975). Decline of maternal behavior in the virgin and lactating rat. J. Comp. Physiol. PsychoL, 89: 722-732. Rosenblatt, J . S. (1965). The basis of synchrony in the behavioral interaction between the mother and her offspring in the laboratory rat. In B. €1. Foss (Ed.), Determinants of Infant Behmioitr. III. London: Methuen. Pp. 3-45, Shoemaker, W. J., and Wurtman, R. J , (1 971). Perinatal undernutrition: Accumulation of uatecholamines in rat brain. Science, 171: 1017-1019. Smart, J. L., and Preece, J. (1973). Maternal behaviour of undernourished mother rats. 4nim. Rehav., 21: 613-619. Smotherman, W. P., Bell, R. W., Starzec, J . , Elias, J., and Zachman, T. (1974). Maternal responses t o incant vocalizations and olfactory cues in rats and mice. Behav. Biol., 1 2 : 55-66. Stern, J. M., and Levin, R. (1976). Food availability as a determinant of the rats' circadian rhythm in maternal behavior. Dev. Psychohiol., 9 : 137-148. Thoman, E. B., and Levine, S. (1970). Effects of adrenalectoniy o n maternal behavior in rats. Dev. Psychobiol., 3 : 237-244. Thoman, E. B., Sproul, M., Seeler, B., and Levine, S. (1970). Influence of adrenalectornv in female rats o n reproductive processes including effects on the foetus and offspring. J. Fndocrinol., 46: 297-303. Winer, S. J. (1971). Statistical Principles in Experimental Design. 2nd Edition. New York: iMcGrawHill. Winick, M.. and Coornbs, J . (1972). Nutrition, environment, and behavioral development. Ann. Rev. hled., 23: 149-1 60.
Recently, a number of reviews of the effects of perinatal malnutrition on later behavior in rodents have pointed out that the laboratory methods used to produce early malnutrition are confounded by variations in other environmental factors, such as amount of early handling or disruption of the mother-infant relationship (Levine & Wiener, 1975; Winick & Coombs, 1972). Because changes in these environmental factors, independent of any change in the nutritional state of the neonate, can produce long-lasting behavioral differences in adulthood (Daly, 1973 ; Denenberg, 1969 ; Newton & Levine, 1968), these nonnutritional variables must be carefully controlled so that effects of perinatal malnutrition can be properly evaluated. Several of the methods commonly used to produce malnutrition in rodents (e.g., restricting the time pups spend with the mother, increasing the litter size to 15-20 pups) produce obvious disruptions in the mother-infant relationship (see reviews by Levine & Wiener, 1975; Plaut, 1970). Another technique, restricting the total food intake of the lactating mother by feeding 50% of normal intake once a day, produces deficits in maternal retrieval behavior and shifts the normal circadian rhythm of maternal care (Smart & Preece, 1973; Stern & Levin, 1976).
Reprint requests should be sent to Dr. Sandra G. Wiener, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305. Received for publication: 19 April 1976 Revised for publication: 14 June 1976 Developmental Psychobiology, 10(3): 243-254 (1 977) @ 1977 by John Wiley & Sons, Inc.
243
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Recent investigations of the maternal behavior of mothers fed a low-protein diet ad libitum (Frahkova, 1971, 1974; Massaro, Levitsky, & Barnes, 1974) have demonstrated both qualitative and quantitative changes in maternal behavior. Fraiikova reported that during 3-min tests on Days 6, 8, and 10 postpartum low-protein mothers retrieve fewer pups with a prolonged latency t o retrieve the first pup. Further, Frahkova observed that low-protein mothers spend less time with their litter and explore more during retrieval tests. On the basis of these observed decreases in 3, puporientcd behaviors, she concluded that low-protein mothers display a deficit in maternal attention toward their litters. On the other hand, Massaro e t al. reported that when undisturbed low-protein mothers are observed by photographic means during the 12-hr dark period, they spend more time with their young than similarly observed control dams. These investigators attributed the inconsistency of these latter observations with those reported by Frafikova to discrepancies in methodology. Together, these studies raise the questions whether low-protein mothers tested for retrieval display an increased amount of time with their young during periods not associated with the retrieval test period and whether the daily test procedures result in an overall decrease in maternal attention toward the litter. The following studies were undertaken t o characterize the low-protein mother’s retrieval behavior and nest-building ability, and to assess the time spent with young during periods not associated with the retrieval/nest-building tests. Whether alterations in the low-protein dam’s maternal behavior are due to her own malnourished condition or her raising malnourished pups was also investigated.
Experiment 1 The purpose of this experiment was t o assess retrieval, nest-building, and contact with offspring during periods not associated with the retrieval test for mothers fed a low-protein diet and to compare these behaviors with those of mothers fed a control diet. Additional sets of mothers not tested for retrieval or nest-building ability were observed for contact with young to control for any disruption of normal mother-infant relationship resulting from these tests.
Subjects Female Sprague-Dawley rats (Rattus nowegicus) received from Simonsen Laboratories (Gilroy, California) at approximately 75 days o f age were housed in groups of 3 in metal pans (28 x 48 x 1 5 cm). Seven days after their arrival, they were mated with Long-Evans males (Simonsen Laboratories, Gilroy, California) t o obtain a genetic hybrid offspring. Daily vaginal smears were taken and the presence of sperm marked the 1st day, Day 0, of gestation. On the 14th day of gestation, females were individually housed in clear plastic cages (28 x 48 x 24 cm) that had a flooring of wood shavings and a grid top that held food pellets and a water bottle. Whereas half of the females (n = 16) continued to be maintained on the stock laboratory diet until weaning (23% protein diet; Feedstuffs Processing Co., San Francisco, California), the other half (n = 16) were placed on a low-protein, isocaloric diet (85%protein diet; Teklad Mills, Madison, Wisconsin),
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on which they were maintained until weaning. On the 20th day of gestation each female was given a packet of newspaper strips for nesting material. Females were checked twice daily for the presence of pups and within 12 hr of parturition (Postpartum Day 0) litters were adjusted to 8 pups (4 males and 4 females). At weaning (Postpartum Day 21), all litters were weighed. Mothers which failed to rear at least 6 pups until weaning were not included in the study. All subjects were kept in an air-conditioned, constant temperature and humidity colony room with controlled lights on a 12-hr on/l2-hr off cycle. Food and water were continuously available. Cages were cleaned at parturition but were subsequently left uncleaned until weaning. Mothers within each nutrition group were assigned alternately to either a group tested for retrieval behavior or a nontested group. This resulted in 4 groups (n = 8 per group): dams fed a low-protein diet and tested for retrieval behavior (LP-T); dams fed a low-protein diet and not tested for retrieval behavior (LP-NT); dams fed a control diet and tested for retrieval (C-T); and dams fed a control diet and not tested for retrieval (C-NT).
Met hods Each day mothers in the LP-T and C-T groups were tested for retrieval behavior at 1% hr before lights-on. For these tests the maternity cages were quietly moved from the colony room to an adjacent anteroom. Both rooms were illuminated by dim red light that remained on only during the testing period t o facilitate observations. In the anteroom dams were placed in holding pans for 2-3 min while their young were removed from the nest and placed in a plastic holding container. The young were then scattered over the floor of the maternity cage opposite the nest. Mothers were then returned to their nest and retrieval of each pup was noted. These observations were continued until the entire litter was retrieved or until 10 min had elapsed. At the end of the retrieval test, the nesting material (paper strips) was removed from the maternity cages and replaced with a fresh packet of nesting material. Cages were then returned to the colony room. At 1 hr and 5 hr after placement of fresh nesting material, cages were examined for the presence of a nest. An animal was scored as having a nest if the paper strips were piled in a circular or semicircular mound to a height of at least 4-5 cm. Maternal contact with young was assessed twice daily in both retrieval tested and nontested groups. These assessments were made once during the dark period under dim red illumination (4 hr before lights-on) and once during the light period (4 hr after lights-on). Each testing period was 30 min long with 15 observations (1 every 2 min) of each mother’s behavior. During each check a female was scored as being in contact with her litter if she was in the nest area with at least 4 pups. Observations of retrieval, nest-building, and maternal contact with young were carried out on Days 2 through 12 postpartum. The decline in the observed maternal retrieval and nest-building behavior and the concomitant increase in offspring activity prevented reliable observations after the 2nd week postpartum. Although the untransformed data are presented in the figures, the frequency of occurrence for the various categories of maternal behavior were subjected to arcsin
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transformations before analysis. The transformed scores for retrieval were analyzed by a 2 (Maternal Nutrition) x 1 1 (Days Postpartum) analysis of variance with the Days factor treated as a within subjects factor. The nest-building data were subjected t o a 2 (Maternal Nutrition) x 11 (Days) x 2 (Time of Observation) analysis of variance with the last 2 factors treated as within subjects factors. Maternal contact was analyzed by a 2 (Maternal Nutrition) x 2 (Tested/Nontested) x 11 (Days) x 2 (Time of Observation) analysis of variance with the last 2 factors treated as within subjects factors.
Results and Discussion The offspring of LP mothers weighed significantly less at weaning than the young of C mothers ( F =532.02, df = 1/288, p < ,001). These body weights (LP offspring: 17.38 2 1.00 g; C offspring: 46.88 ? .84 g) are similar t o those reported for pups raised b p mothers fed a low-protein diet or control diet during lactation (Massaro et al., 1974; Shoemaker & Wurtman, 1971). Summed observations over days showed that LP mothers retrieved fewer pups during a 10-niin retrieval test when compared t o C mothers ( F = 4.37, df = 1/14, I J < .05; Fig. 1). However, the significant Maternal Nutrition x Days interaction (I; = 2.34, df = 10/140, p < .02) indicated that although the retrieval behavior of LP mothers did not differ from C mothers during the early days postpartum, these LP mothers retrieved a significantly smaller percentage of their litter ( p < . O l ) beginning on Day 9 postpartum (post hoc tests for simple main effects; Winer, 1971, p. 347). Thus, LP mothers showed a deficit in this measure of retrieval behavior which became apparent only after the first week postpartum.
Fig 1 . Daily percentage of litter retrieved by control (C) and low-protein (LP) mothers during a l0-niin retrieval test on Days 2-1 2 postpartum (C > LP on Days 9-1 2).
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Fig. 2. Percentage of control (C) and low-protein (LP) mothers with nests within 1 hr and 5 hr of receiving nestbuilding material (X f S.E.).
A significant Maternal Nutrition x Time of Observation interaction ( F = 24.08, d f = 1/28, p < .001) was found in the nest-building behavior of LP and C mothers (see Fig. 2). Further analysis of this effect demonstrated that LP mothers built fewer nests than controls 1 hr after being presented with a new packet of nesting material ( p < .OS) but did not differ from controls at 5 hr. Thus, the disruption in nest-building behavior appeared to be transient reflecting, perhaps, the rate not the ability of low-protein mothers to construct a nest. Analyses of the mothers' behavior during the 2 daily observation periods yielded a number of significant effects. As previously reported by Grota and Ader (1969), mothers spend more time with their litters during the lights-on period and the significant Time of Observation effect bears this out ( F = 128.95, df = 1/28, p < .001). However, because Time of Observation did not interact with Maternal Nutrition or Days, only the average daily score is reported here. The main effect of Maternal Nutrition ( F = 65.06, df = 1/28, p < .OOl) and the significant Maternal Nutrition x Days interaction ( F = 3.30, df = 10/28. p < .Ol) indicate that the LP mothers spent more time in contact with their offspring on postpartum Days 3-12 (see Fig. 3). This was the case whether or not the mother was subjected to a daily retrieval/nest-building test ( F = 1.64, d f = 1/28, p > .05). Whereas the C mothers showed a characteristic decrease in time spent with young as the pups grew older (Rosenblatt, 1965), the LP mothers maintained a higher level of maternal contact during these days.
Experiment 2 Although Experiment 1 demonstrated alterations in the maternal behavior of low-protein mothers, it did not indicate whether these alterations were due t o the
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mother’s malnourished condition or the size and stimulus characteristics of the pups she reared. Rodent offspring are n o t passive recipients of tnaternal care. but provide cues that organize and coordinate mother-infant behavior sequences (Noirot, 1972; Reisbick, Rosenblatt, & Mayer, 1975; Smotherman, Bell, Starzec, Elias, & Zachman, 1974). Possibly, the increased time low-protein mothers spend with young may be elicited by the small size of her pups, which maintain an earlier postpartum form of maternal behavior characterized by more maternal contact (Rosenblatt, 1965). However, if the stimulus characteristics of the growth-stunted pups are the only factor, we would expect that these characteristics would also elicit an adequate pattern of retrieval and nest-building. Obviously, this is not the case because the low-protein mothers display a deficit in these 2 behaviors. Thus. the mother’s nutritional state must also play an important role in determining the alterations in these maternal behaviors. The purpose of Experiment 2 was t o compare the maternal behavior of lowprotein mothers t o the behavior of another type of mother that also raises growthstunted pups: an adrenalectoniized mother. Adrenalectomy was chosen as a preparation because of previous work demonstrating that adrenalectomized mothers rear offspring of similar body weight t o those reared b y low-protein mothers (Thoman, Sproul, Seeler. & Levine, 1970). Although the absence of adrenal glucocorticoids is resporisible for the reduction in lactation (Cowie & Folley, 1947), the presence of a functioning adrenal is not necessary for the initiation or maintenance of maternal responsiveness in rats (Thoman & Levine, 1970). Adrenalectomized mothers deliver young, show maternal behavior, and rear viable offspring.
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Subjects The subjects of this experiment were Sprague-Dawley female rats obtained, housed, mated, and rehoused in the same manner as in Experiment 1. In addition to the low-protein (LP) and control (C) diet mothers, a set of mothers that were adrenalectornized (ADX) were studied. On Day 16-17 of gestation, mothers assigned t o the ADX group underwent surgery under ethyl-ether anesthesia. The animal's back was shaved, and a 4-cm dorsal incision was made. The peritoneum on the right side was then punctured at the posterior end of the rib cage, and a retractor was inserted to permit exposure of the adrenal gland. The adrenal was excised with curved forceps and examined t o assure that it had not been ruptured. If damage to the adrenal had occurred, the animal was discarded. This procedure was then carried out on the animal's left side, after which the peritoneum openings were closed with 5-0 silk suture. Wound clips were used t o close the mother's back and penicillin (Bicillin, Wyeth) was administered. Salt licks were provided to compensate for the lack of salt regulation resulting from adrenalectomy. Completeness of adrenalectomy was determined by plasma corticosteroid levels after ether stress on Day 14 postpartum and females showing significant steroid elevations were not included in the analyses of maternal behavior. At parturition, mothers within LP, C, and ADX groups were divided into groups tested for retrieval behavior (LP-T, C-T, ADX-T [n = 7 per group]) or nontested groups (LP-NT, C-NT, ADX-NT [n = 7 per group] ).
Methods The procedures used to test retrieval, nest-building, and maternal contact with young were identical to those used in Experiment 1. Body weights of the pups from LP-T, C-T, and ADX-T mothers were recorded daily just prior t o the retrieval testing. These weights were analyzed by a 2-factor, 3 (Offspring Type) x 11 (Days of Age), analysis of variance with the Days factor treated as a within subjects factor. The maternal behavior data were analyzed as described in Experiment 1, with the addition of the 3rd maternal group (ADX). Tests on simple main effects and subsequent comparisons by Newman-Keuls procedures were carried out where appropriate.
Results and Discussion The main effect of Offspring Type ( F = 73.86, df = 2/18, p < .OOl) and the interaction of Offspring Type x Age ( F = 72.71, df = 20/180, p < .001) reflect the different growth rates for each Offspring Type (see Fig. 4). Tests for simple effects of Offspring Type a t each age yielded the following results: progeny of LP and ADX mothers weighed less than controls on Days 3-12 (p < .Ol), whereas the offspring of ADX mothers did not begin to differ from LP young until Day 9 (p < .05). Thus, the LP and ADX mothers raised pups of similar size (within 2-3 g) throughout the entire testing period.
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The main effect of Maternal Group (F = 5.30, d f = 2/18, p < .02) and the Materd f = 20/180, p < ,001) indicate, as in Experiment 1 , that the retrieval performance of LP mothers differed froin that of the controls (see Fig. 5). In this experiment, however, differences were already present on Day 6 postpartum ( p < .0S), On the other hand, ADX mothers did not demonstrate the decline in retrieval behavior characteristic of the LP mothers. The maintenance of normal retrieval behavior in ADX mothers confirms and extends the previous report by Thoman and Levine (1970). The nest-building behavior of ADX mothers was indistinguishable froin controls at both the 1-hr and 5-hr intervals (see Fig. 6). Again the LP mothers showed deficits in the number of nests constructed within 1 hr after presentation of nesting materials, which resulted in the significant Maternal Group x Timc of Observation interaction ( F = 35.85, d f = 2/36, p < ,001). Thus, the disruption of retrieval and nest-building behavior in the LP mothers appears not to be due only t o the size of her offspring, but may be a direct effect of the mother’s nutrition on her maternal behavior. The ADX mothers, raising similar size pups, did not display retrieval or nest -building deficits. The analysis of maternal contact with young indicated a main effect of Maternal Group ( F = 45.42, d f = 2 / 3 6 , p < .001); however, the Maternal Group x Days interaction iial Group x Days interaction ( F = 2.89,
MOTHERS REARING MALNOURISHED OFFSPRING
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Fig. 5. Daily percentage of litter retrieved by control (C), low-protein (LP), and adrenalectomized (ADX) mothers during a 10-min retrieval test on Days 2-12 postpartum (C > LP on Days 6-12; ADX > LP on Days 8-12).
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Fig. 6 . Percentage of control (C), low-protein (LP), and adrenalectomized (ADX) mothers with i S.E.). nests within 1 hr and 5 hr of receiving nest-building material
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Fig. 7 . Daily mean percent of time control (C), low-protein (LP), and adrenalectonlized (ADX) mothers were observed in contact with litter during two 30-min observation periods o n Days 2-12 postpartum (C < LP, ADX on Days 4-1 2).
was also significant ( F = 3.31, d f = 20/360, p < .01; Fig. 7). In contrast with the results for retrieval and nest-building, the ADX mothers resembled LP mothers on the measure of contact with young. Both LP and ADX mothers showed an increase in the amount of time spent with young when compared t o C mothers on Days 4-12 postpartum O-, < .05). Thus, unlike retrieval and nest-building, contact with young appears t o be related to the stimulus qualities of the pups (e.g., their small size, suckling characteristics, ultrasonic vocalizations, or lack of mobility due to retarded neuromotor development).
General Discussion In this report we have demonstrated that mothers fed a low-protein diet exhibit deficits in retrieval during the 2nd week of lactation, a deficit in their rate of building a nest, and an increase in time spent with young. These results not only confirm the results of Frahkovi (1971, 1974) and Massaro et al. (1974), but also demonstrate that these alterations in various measures of maternal behavior occurred concomitantly. The low-protein mother may under certain circumstances (e.g., during a retrieval test) display decreased attention toward her offspring; however, this does not generalize t o an overall decrease in time spent with her pups during those periods when she is relatively undisturbed by the experimenter. The comparison of the maternal behavior of the low-protein and adrenalectoniized dams suggests that both the mother's nutritional status and her pup's malnutrition play a role in determining the material behavior of the low-protein mother. On the
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one hand, although the adrenalectomized dams raised pups of similar size, they did not display the deficits in retrieval or nest-building of low-protein mothers. This finding suggests that the malnourished condition of the low-protein mother is at least partly responsible for these deficits. Whether malnutrition directly affects retrieval and nest-building or whether these alterations reflect a more general susceptibility to disturbance in their environment remains to be determined. Support for the latter is provided by the appearance of retrieval deficits only after repeated testing (during the 2nd week of lactation). These experiments leave the question unanswered whether the repeated testing and the disturbance accompanying the daily retrieval and nestbuilding test procedure are responsible for the decline in retrieval or whether the decline occurs on Days 6-8 regardless of past testing experience. On the other hand, the stimulus characteristics of the pups appear to be the important factor in eliciting an increase in maternal contact in that both adrenalectomized and low-protein mothers displayed this alteration in maternal behavior. A recent report (Lynch, 1976) on a nipple-ligation method to produce malnourished offspring has indicated that this type of mother raising growth-stunted young spends an increased amount of time with her litter. Thus, any preparation used to produce perinatal malnutrition in rodent offspring will probably induce mothers to spend increased time with young unless some other behavior competes for the mother’s attention. This would probably account for the decreased time spent with young observed in the intake-restricted mother (Smart & Preece, 1973). These restricted mothers spent less time with their offspring compared t o nonrestricted mothers during observations preceding the daily food presentation, but spent more time with the litter during the succeeding periods. The importance of these alterations in the low-protein dam’s maternal behavior in relation to the later behavioral development of the offspring is yet to be determined. However, if the alterations we have shown in the mother’s behavior result in a disruption of the mother-infant relationship, then any long-term influence on the pup’s development attributed directly to early malnutrition must be viewed with caution. These effects could as easily be attributed to other demonstrable influences of early experience on later behavior.
Notes This research was supported by Biosciences Training Grant MH-8304 from t h e National Institute of Mental Health (to S. G. Wiener, R. Levin, and W. P. Smotherman), Research Grant NICH&HD-02881 from the National Institutes of Health, and USPHS Research Scientist Award K5MH-19936 from the National Institute of Mental Health (S. Levine). S. Wiener is now at The Salk Institute, San Diego, CA.
References Cowie, A. T., and Folley, S. I. (1947). Adrenalectomy and replacement therapy in lactating rats. 2 . Effects of deoxycorticosterone acetate o n lactation in adrenalectomized rats. J. Endocrind, 5: 14-23. Daly, M. (1973). Early stimulation of rodents: A critical review of present interpretations. Brit. J. P S J J C ~ 64: O ~ .435-460. ,
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Denenberg, V. H. (1969). The effects of early experience. In E. S. E. Hafez (Ed.), The Behaviour of Domestic Animals. London: Bailliere, T i n d a l l k Cassell. Pp. 109-138. I:rafikovi, S. (1971). Relationship between nutrition during lactation and maternal behaviour of rats. Activ. nerv. sup., 13: 1-8. Frahkovi, S. (1974). Effects of protein deficiency in early life and during lactation on maternal behaviour. Baroda J. Nutr.. I : 21-28. Grata, L. J., and Ader, R. (1969). Continuous recording of maternal behaviour in Kattus norvegfcus. Anim. Behav., 1 7 : 722-729. Levine, S., and Wiener, S. (1975). A critical analysis of data o n malnutrition and behavioral deficits. Adv. Pediat., 22: 11 3-1 36. Lynch, A. (1976). Postnatal undernutrition: An alternative method. Del,. Psychohiol., 9 : 39-48. Massaro, T . F., Levitsky, D. A,, and Barnes, A. H. (1974). Protein malnutrition in t h e rat: Its et'fects on maternal behavior and pup development. Dev. Psychohiol., 7: 551-561. Newton, G., and Levine, S. (1968). Early Experience and Behavior. Springfield, Ill.: C. C. Thomas. Noirot, E. (1972). The onset of maternal behavior in rats, hamsters, and mice. A selective review. Adv. Study Behav., 4 : 107-145. Plaut, S. M. (1970). Studies of undernutrition in the young rat: Methodologiwl considerations. Dev. Psychobiol., 3 : 157-167. Keisbick, S., Rosenblatt, J. S., and Mayer, A . D. (1975). Decline of maternal behavior in the virgin and lactating rat. J. Comp. Physiol. PsychoL, 89: 722-732. Rosenblatt, J . S. (1965). The basis of synchrony in the behavioral interaction between the mother and her offspring in the laboratory rat. In B. €1. Foss (Ed.), Determinants of Infant Behmioitr. III. London: Methuen. Pp. 3-45, Shoemaker, W. J., and Wurtman, R. J , (1 971). Perinatal undernutrition: Accumulation of uatecholamines in rat brain. Science, 171: 1017-1019. Smart, J. L., and Preece, J. (1973). Maternal behaviour of undernourished mother rats. 4nim. Rehav., 21: 613-619. Smotherman, W. P., Bell, R. W., Starzec, J . , Elias, J., and Zachman, T. (1974). Maternal responses t o incant vocalizations and olfactory cues in rats and mice. Behav. Biol., 1 2 : 55-66. Stern, J. M., and Levin, R. (1976). Food availability as a determinant of the rats' circadian rhythm in maternal behavior. Dev. Psychohiol., 9 : 137-148. Thoman, E. B., and Levine, S. (1970). Effects of adrenalectoniy o n maternal behavior in rats. Dev. Psychobiol., 3 : 237-244. Thoman, E. B., Sproul, M., Seeler, B., and Levine, S. (1970). Influence of adrenalectornv in female rats o n reproductive processes including effects on the foetus and offspring. J. Fndocrinol., 46: 297-303. Winer, S. J. (1971). Statistical Principles in Experimental Design. 2nd Edition. New York: iMcGrawHill. Winick, M.. and Coornbs, J . (1972). Nutrition, environment, and behavioral development. Ann. Rev. hled., 23: 149-1 60.
