Protein Malnutrition Induced During Gestation: Its Effect on Pup Development and Maternal Behavior THOMAS F. MASSARO Division of Nutritional Sciences DAVID A. LEVITSKY Division of Nutritional Sciences and Department o f Psychology RICHARD H. BARNES Division of Nutritional Sciences Cornell University Ithacn, New York
Repeated time-lapse photographic observations were used t o examine the ontogeny of behavioral development in prenatally malnourished rat pups following birth. Pups born t o dams receiving a low protein diet (7% casein by weight) were fostered at birth to well-nourished dams and behavioral observations were made at 4-day intervals. Dams nursing gestationally malnourished pups were observed t o spend more time in the nesting area with their pups towards the end of lactation. Moreover, the behavioral development (locomotion, feeding, and rearing and climbing activity) of gestationally malnourished offspring was depressed when compared t o their controls. The results indicate that prenatal undernutrition alters the behavioral development of the offspring and maternal behavior of the dam in a manner which reduces the exposure of the pup to the immediate environment.
Undernutrition restricted to prenatal development in the rat has been shown to produce long-term alterations in behavior (Hsueh, Simonson, Chow, & Hanson, 1974; Smart, Dobbing, Adlard, Lynch, & Sands, 1973). The mechanism(s), whether organismic or behavioral, which may account for these disturbances are poorly understood. One possibility, however, is that the effects of prenatal malnutrition may occur through alteration in the early postnatal environment of the suckling young. Levitsky and Barnes (1972, 1973) have suggested that undernutrition during the early postnatal period of development functionally restricts the young organism from certain aspects Reprint requests should be sent to Dr. Thomas F. Massaro, Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, New York 14853, U.S.A. Received for publication 14 April 1976 Revised for publication 28 July 1976 Developmental Psychobiology, 1O(4): 339-345 ( 1977) @ 1977 by John Wiley & Sons, Inc.
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of its environment and that a disturbance in the learning experience during this period may account for many of the long-term behavioral effects. Massaro, Levitsky, and Barnes (1974) have clearly demonstrated the effect of postnatal malnutrition in reducing the development of behaviors necessary for environmental exploration. A similar disturbance may be produced in the prenatally malnourished offspring through delaying both physical and behavioral maturation during the postnatal period of development. In mice malnourished during gestation, Bush and Leathwood (1975) observed a significant depression in the ontogeny of various psychomotor reflexes following birth. The data in the rat are more equivocal although a tendency toward delayed psychomotor development has been observed (Simonson, Sherwin, Anilane, Yu, & Chow, 1968; Smart & Dobbing, 1971). The present investigation was therefore undertaken t o observe systematically the nlanner in which prenatal malnutrition affects behavioral development of the rat pup during postnatal development. Emphasis was directed at determining to what extent those pup behaviors involved in environmental exploration are altered as a consequence of prenatal malnutrition.
Subjcc:t s Adult virgin rats (Rattus norwegicus) of the Sprague-Dawley-derived Holt~inan strain weighing approximately 240 g were mated in our laboratory. Breedinp was staggered such that 4-6 dams were mated every day for 4 days t o facilitate behavioral observations. Thereafter, sperm positive females were randomly assigned t o 1 of 2 dietary treatments. The dams were fed ad lihitunz tlirougliout gestation either the control diet (25% casein) o r an isocaloric low protein diet ( 7 9 casein) described in Barnes, Kwong, Morrissey, Vilhjalmsadottir, and Levitsky (1 973). All animals were housed individually in standard laboratory cages for the first 10 days of gestation and were then transferred to Plexiglas maternity cages (30 x 30 x 30 cni) for the remainder of gestation. Nesting material (wood shavings) was provided t o parturition and throughout lactation. Lighting was maintained on a 12-hr light: 12-hr dark cycle with lights on at 0600 hours. Body weight recordings of the dams were made on Days 2 (day sperm detected). 7, 14, and just prior to parturition.
Procedure Within 3 h r following birth pups were removed from each dam, individually weighed, and sexed, and the number of pups in each litter was reduced t o 8 of which a least 5 were males. Both the control (C) and gestationally malnourished (DG) pups were fostered to other dams, all of which received the control diet throughout gestation. Through the 29 days of lactation all dams were fed the control diet (25% casein) ad lib. Pups were handled only for weighing purposes and for necessary pup replacement of both C and DG pups in order to maintain the litter size at 8. Seven control litters and 6 litters of gestationally malnourished pups were maintained during lactation. Replacement stocks of both C and DG pups were also maintained throughout the experiment.
Photographic Observations and Behavioral Classification The method of photographic observation and the classification of behavior of the dams and pups during the lactation period have been described previously (see Massaro,
MALNUTRITION AND BEHAVIOR IN THE RAT
341
Levitsky, & Barnes [ 19741 for details). Repeated time-lapse photographic observations were carried out during the 12-hr dark cycle on Days 1 (day of delivery), 5 , 9, 13, 17, 21, 25, and 29 of lactation. Data were treated statistically by Student's t test and by analyses of variance employing a mixed effect model with repeated measures on time (Winer, 1962). Nonparametric analysis was performed with the Wilcoxon Rank Sum Test.
Results Body Weights The rate of increase in weight during gestation was less in the experimental dams (DG) as shown by a significant Diet x Time interaction ( F = 34.74, df = 3/66. p < .01; see Fig. 1). At birth pups born to malnourished dams weighed significantly less (mean wt. = 5.51 g) than their controls (mean wt. = 6.63 g; t = 5.05, df = 38, p < .001). This difference in body weight was maintained. throughout lactation ( F = 2.28, df = 7/77, p < .OS). At weaning body weights of DC pups were 93% of control weights. Compared t o their controls offspring of undernourished mothers evidenced a greater incidence of postnatal mortality (t = 2.42, df = 11, p < .05).
Dam Behavior The behavior of dams nursing experimental (DC) or control (C) pups was similar throughout the major portion of lactation. An analysis of variance failed to reveal DAM WEIGHT
I
1 1
7
rl
11
davs, gestation
Fig. 1. Mean body weight of dams maintained o n a 25% casein (control) or 7% casein (experimentd) diet during gestation. (Vertical bars indicate S.E.)
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MASSARO, LEVITSKY, AND BARNES
statistically significant differences between the dams for the C and DG pups in the frequency of observed self-grooming behavior (F = 1.19, df = 7/77, p > .OS), feeding ( F = .70, d f = 7/77), rearing (support hind legs only) (F = 1.31, d f = 7/77, p > .05), or locomotor behavior ( F = .46, df = 7/77) throughout lactation. Differences were not detected in the frequency of observing the dams in the nesting area with the pups up t o Day 21 (see Fig. 2). An analysis of variance did, however. reveal a significant Diet x Time interaction (F = 3.15, df = 7/77, p < .01). As shown by a Wilcoxon Rank Sum Test, the frequency of observing this behavior was significantly greater (p < . O l ) in those dams nursing gestationally malnourished pups only on Days 2 5 and 29.
Pup Behavior Litter fragmentation increased in both groups throughout the preweaning period of development (see Fig. 3). The rate of development of this behavior was greater for the controls. as evidenced by a significant Diet x Age interaction (F = 4.72, d f = 4/44, p < .01). The rate of development of self-feeding behavior was less in the experimental group as shown by a significant Diet x Time interaction (F = 4.56, df = 4/44, p < .O I ; see Fig. 3). N o differences were observed in the development of drinking behavior. The rate of development of rearing behavior was less in DG pups as shown by a significant Diet x Time interaction ( F = 4.03, df = 4/44, p < .Ol). Although an analysis of variance failed t o reveal a significant difference between C and DG pups in the development of climbing behavior, Wilcoxon Rank Sum Test proved that DG pups display less climbing activity on Day 29 (p < .05; see Fig. 3).
Discussion The results of the present investigation demonstrate that gestational malnutrition produces a significant effect on behavioral development of the offspring, particularly TIME IN NESTING AREA ~ . _ _
control deficient gestation
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Repeated time-lapse photographic observations were used t o examine the ontogeny of behavioral development in prenatally malnourished rat pups following birth. Pups born t o dams receiving a low protein diet (7% casein by weight) were fostered at birth to well-nourished dams and behavioral observations were made at 4-day intervals. Dams nursing gestationally malnourished pups were observed t o spend more time in the nesting area with their pups towards the end of lactation. Moreover, the behavioral development (locomotion, feeding, and rearing and climbing activity) of gestationally malnourished offspring was depressed when compared t o their controls. The results indicate that prenatal undernutrition alters the behavioral development of the offspring and maternal behavior of the dam in a manner which reduces the exposure of the pup to the immediate environment.
Undernutrition restricted to prenatal development in the rat has been shown to produce long-term alterations in behavior (Hsueh, Simonson, Chow, & Hanson, 1974; Smart, Dobbing, Adlard, Lynch, & Sands, 1973). The mechanism(s), whether organismic or behavioral, which may account for these disturbances are poorly understood. One possibility, however, is that the effects of prenatal malnutrition may occur through alteration in the early postnatal environment of the suckling young. Levitsky and Barnes (1972, 1973) have suggested that undernutrition during the early postnatal period of development functionally restricts the young organism from certain aspects Reprint requests should be sent to Dr. Thomas F. Massaro, Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, New York 14853, U.S.A. Received for publication 14 April 1976 Revised for publication 28 July 1976 Developmental Psychobiology, 1O(4): 339-345 ( 1977) @ 1977 by John Wiley & Sons, Inc.
339
340
MASSAKO, LEVITSKY, AND BARNES
of its environment and that a disturbance in the learning experience during this period may account for many of the long-term behavioral effects. Massaro, Levitsky, and Barnes (1974) have clearly demonstrated the effect of postnatal malnutrition in reducing the development of behaviors necessary for environmental exploration. A similar disturbance may be produced in the prenatally malnourished offspring through delaying both physical and behavioral maturation during the postnatal period of development. In mice malnourished during gestation, Bush and Leathwood (1975) observed a significant depression in the ontogeny of various psychomotor reflexes following birth. The data in the rat are more equivocal although a tendency toward delayed psychomotor development has been observed (Simonson, Sherwin, Anilane, Yu, & Chow, 1968; Smart & Dobbing, 1971). The present investigation was therefore undertaken t o observe systematically the nlanner in which prenatal malnutrition affects behavioral development of the rat pup during postnatal development. Emphasis was directed at determining to what extent those pup behaviors involved in environmental exploration are altered as a consequence of prenatal malnutrition.
Subjcc:t s Adult virgin rats (Rattus norwegicus) of the Sprague-Dawley-derived Holt~inan strain weighing approximately 240 g were mated in our laboratory. Breedinp was staggered such that 4-6 dams were mated every day for 4 days t o facilitate behavioral observations. Thereafter, sperm positive females were randomly assigned t o 1 of 2 dietary treatments. The dams were fed ad lihitunz tlirougliout gestation either the control diet (25% casein) o r an isocaloric low protein diet ( 7 9 casein) described in Barnes, Kwong, Morrissey, Vilhjalmsadottir, and Levitsky (1 973). All animals were housed individually in standard laboratory cages for the first 10 days of gestation and were then transferred to Plexiglas maternity cages (30 x 30 x 30 cni) for the remainder of gestation. Nesting material (wood shavings) was provided t o parturition and throughout lactation. Lighting was maintained on a 12-hr light: 12-hr dark cycle with lights on at 0600 hours. Body weight recordings of the dams were made on Days 2 (day sperm detected). 7, 14, and just prior to parturition.
Procedure Within 3 h r following birth pups were removed from each dam, individually weighed, and sexed, and the number of pups in each litter was reduced t o 8 of which a least 5 were males. Both the control (C) and gestationally malnourished (DG) pups were fostered to other dams, all of which received the control diet throughout gestation. Through the 29 days of lactation all dams were fed the control diet (25% casein) ad lib. Pups were handled only for weighing purposes and for necessary pup replacement of both C and DG pups in order to maintain the litter size at 8. Seven control litters and 6 litters of gestationally malnourished pups were maintained during lactation. Replacement stocks of both C and DG pups were also maintained throughout the experiment.
Photographic Observations and Behavioral Classification The method of photographic observation and the classification of behavior of the dams and pups during the lactation period have been described previously (see Massaro,
MALNUTRITION AND BEHAVIOR IN THE RAT
341
Levitsky, & Barnes [ 19741 for details). Repeated time-lapse photographic observations were carried out during the 12-hr dark cycle on Days 1 (day of delivery), 5 , 9, 13, 17, 21, 25, and 29 of lactation. Data were treated statistically by Student's t test and by analyses of variance employing a mixed effect model with repeated measures on time (Winer, 1962). Nonparametric analysis was performed with the Wilcoxon Rank Sum Test.
Results Body Weights The rate of increase in weight during gestation was less in the experimental dams (DG) as shown by a significant Diet x Time interaction ( F = 34.74, df = 3/66. p < .01; see Fig. 1). At birth pups born to malnourished dams weighed significantly less (mean wt. = 5.51 g) than their controls (mean wt. = 6.63 g; t = 5.05, df = 38, p < .001). This difference in body weight was maintained. throughout lactation ( F = 2.28, df = 7/77, p < .OS). At weaning body weights of DC pups were 93% of control weights. Compared t o their controls offspring of undernourished mothers evidenced a greater incidence of postnatal mortality (t = 2.42, df = 11, p < .05).
Dam Behavior The behavior of dams nursing experimental (DC) or control (C) pups was similar throughout the major portion of lactation. An analysis of variance failed to reveal DAM WEIGHT
I
1 1
7
rl
11
davs, gestation
Fig. 1. Mean body weight of dams maintained o n a 25% casein (control) or 7% casein (experimentd) diet during gestation. (Vertical bars indicate S.E.)
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MASSARO, LEVITSKY, AND BARNES
statistically significant differences between the dams for the C and DG pups in the frequency of observed self-grooming behavior (F = 1.19, df = 7/77, p > .OS), feeding ( F = .70, d f = 7/77), rearing (support hind legs only) (F = 1.31, d f = 7/77, p > .05), or locomotor behavior ( F = .46, df = 7/77) throughout lactation. Differences were not detected in the frequency of observing the dams in the nesting area with the pups up t o Day 21 (see Fig. 2). An analysis of variance did, however. reveal a significant Diet x Time interaction (F = 3.15, df = 7/77, p < .01). As shown by a Wilcoxon Rank Sum Test, the frequency of observing this behavior was significantly greater (p < . O l ) in those dams nursing gestationally malnourished pups only on Days 2 5 and 29.
Pup Behavior Litter fragmentation increased in both groups throughout the preweaning period of development (see Fig. 3). The rate of development of this behavior was greater for the controls. as evidenced by a significant Diet x Age interaction (F = 4.72, d f = 4/44, p < .01). The rate of development of self-feeding behavior was less in the experimental group as shown by a significant Diet x Time interaction (F = 4.56, df = 4/44, p < .O I ; see Fig. 3). N o differences were observed in the development of drinking behavior. The rate of development of rearing behavior was less in DG pups as shown by a significant Diet x Time interaction ( F = 4.03, df = 4/44, p < .Ol). Although an analysis of variance failed t o reveal a significant difference between C and DG pups in the development of climbing behavior, Wilcoxon Rank Sum Test proved that DG pups display less climbing activity on Day 29 (p < .05; see Fig. 3).
Discussion The results of the present investigation demonstrate that gestational malnutrition produces a significant effect on behavioral development of the offspring, particularly TIME IN NESTING AREA ~ . _ _
control deficient gestation
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