Brain Research Bulletin, All rights of reproduction
Vol. 2, pp. 223-229, 1977. Copyright 0 ANKHO in any form reserved. Printed in the U.S.A.
International
Inc.
Feeding and Drinking in Rats Maintained on a Low Protein Diet’” JOHN E. JALOWIEC3, JANE A. CHISHOLM, WILLIAM B. FORBES, PETER J. MORGANE AND OSCAR RESNICK Worcester Foundation for Ejcperinlental Biology, 222 Maple Avenue, ~~rews~~r~, MA 01545 (Received 22 April 1977) JALOWIEC, J. E., J. A. CHISHOLM, W. B. FORBES, P. J. MORGANE AND 0. RESNICK. Feed&g and dr~nk~lzgin rats ~ajntajned on a low profein diet. BRAIN RES. BULL. Z(3) 223-229, 1977. - Male and female rats born of protein malnourished mothers were fed a low-protein diet (8% casein) for 150 days after weaning and daily food and water intakes and body weights were monitored. Although daily intakes of diet throughout the study were significantly lower than those of rats maintained on a normal protein diet (25% casein) or stock diet, intakes/100 g body weight were significantly greater. Daily increments in body weight, as percent of previous day’s weight, were consistently higher in rats fed the low-protein diet in comparison to rats fed the normal protein diet. Marked retardation of body growth was evident throughout the study although feeding efficiency ratios (daily body wt. increment per g daily food intake) were comparable among the various dietary groups. Compensation for reduction of the protein component of the diet by increased daily food intake/100 g body weight did not alleviate growth retardation. Protein malnutrition Feeding and drinking
Growth behavior
retardation
Incremental
body
weight
Feeding
efficiency
lactation, female rats fed on a low-protein diet did not compensate for reduced protein availability by increasing food intake. The fatter finding is somewhat surprising since the often severe and general debilitation resulting from long-term protein malnutrition could be ameliorated by increased ingestion of the deficient diet. Earlier reports by Adolph [l] and Peterson and Baumgardt [ 10,111 have demonstrated that rats reliably compensate for the addition of non-nutritive bulk to their diets by increasing food intake. Although the importance of dietary protein intake in limiting or increasing total energy intake is unclear, it has been documented that rats can recognize and respond differentially to diets containing various amounts of protein [4]. These reports suggest that rats feeding on diets deficient in protein might adjust their food intake to insure adequate nutrition for growth despite dietary deficiencies. In an attempt to closely paraiiel the human condition, in which mothers are often protein malnourished during gestation and lactation, we initiated protein malnutrition by feeding an 8% protein diet to female rats 5 weeks prior to in~pregnat~on and continued this dietary paradigm throughout gestation and lactation as well as throughout the life of the offspring. In view of studies hy Chow and Lee [ 2)) this paradigm should result in maximal differences between malnourished rats and rats fed a diet containing normal amounts of protein (25%).
IN VIEW OF the inherent difficulties in specifying and controlling the variables associated with protein malnutrition in the human condition f 131, animal models, using rats fed diets deficient in protein, but not calories, have become especially important. Various dietary paradigms have been employed ranging from protein deprivation prior to or at the time of weaning to life-long protein deprivation. Although the generality of findings from experiments using rats to the human condition can be readily assumed on the basis of developmental similarities between species [ 31, generality between findings from different laboratories is often tenuous because of the absence of critical normative data describing the patterns of growth and food intake for rats subjected to different degrees and periods of protein deprivation. For example, a model of malnutrition using protein deprivation only before weaning may not be easily compared to a model based on life-long protein deprivation. In addition, the degree of protein deprivation may be modified by significant differences in food intake between normal and malnourished rats. A recent report suggests that female rats fed a low-protein diet compensate for inadequate protein by ingesting more of the low-protein diet, thereby ingesting more calories and more protein during periods of physiological stress such as pregnancy [7]. However, during periods of mature growth or during
‘Supported by a grant from the National Institute of Health (HD06364). ‘Address correspondence to: Dr. Peter J. Morgane, Worcester Foundation for Experimental Biology, ‘Present address: Department of Psychology, Mount Holyoke College, South Hadley, MA, 223
Shrewsbury.
MA (11545
JALOWIEC
224 The present report describes the alterations in food and water intake and body weight of male and female rats born of mothers maintained on a low-protein diet prior to mating and during gestation and lactation, and themselves maintained on the deficient diet for 150 days after weaning. Data from animals fed a standard laboratory diet as well as from rats fed an isocaloric normal protein diet have been included for comparison. METHOD
Five weeks prior to mating, normal female albino rats (Sprague-Dawley strain, Charles River Breeding Laboratories, Wilmington, MA) were ailowed ad lib access to water and ~onlmercially prepared diets (General Bio~hemi~a~s, Chagrin Falls, OH) containing either 8% or 25% casein as the source of protein. An additional group was allowed access to water and powdered standard laboratory diet (Charles River Rat Formula 4RF). The diet compositions for the 2 test diets and Charles River Rat Formula are given in Tables 1, 2, 3, and 4. The salt mix for each of the test diets is that of Rogers and Harper [ 121. The vitamin mix used for each test diet is given in Table 5. The females were mated with normal males and, after delivery of the young, litter size was reduced to 8 and neonates were randomly redistributed among mothers delivering on the same day and fed the same diet. On Day 21 postpartum, 6 males and 6 females from each dietary regimen were weaned and housed individually (cage size - 45 cm x 27 cm x 27 cm, L., W., D.) with free access to water and the diet of their mother.
1
TABLE
PERCENT NUTRITIONAL COMPOSITION OF
Component Protein Fat Carbohydrate Salt Mix Vitamin Mix Water Nonnutritive Filler Kcal/g
DIET 25%’ Casein Riet*
80/c Casein Diet*
Stock Diet*
21.8 15.4 50.9 4.7 1.0 2.2 4.1 4.3
7.0 15.1 67.9 4.7 1.0 0.9 4.2 4.3
26.3 7.1 42.9 3.5 0.3 9.6 10.3 3.0
*These diets were supplemented casein
lacks this essential
DIETS
amino
with L-methionine
(0.4%)
since
acid.
Beginning on the day of weaning and continuing for 150 days, water intakes from calibrated drinking tubes (k 1 .O ml with steel spouts, food intakes (by weighing spillresistant food containers) and body weights (to the nearest gram) were measured. Food cups and water tubes were refilled every other day. Except for the measurement period (9:00- 1 1 :00 a.m.) the animals were left undisturbed in a temperature-controlled (23”) room with a 12 hr/ I 2 hr light/dark cycle. Analyses of the data were carried out with the aid of a general purpose computer (G.E. time sharing). For the purposes of this report, intakes of water and food and daily body weights were grouped into 15 consecutive IO-day
TABLE 25% CASEIN
TEST
2
DIET (TEKLAD RESEARCH TD 73066)
Casein, High Protein L-~~ethionine Sucrose Dextrose, Anhydrous, Technical Dextrin. White, Technical Corn Oil Nonnutritive Fiber (cellulose) Mineral Mix, Rogers-Harper (Cat. No. 170760) Vitamin Mix, Teklad (Cat. NO. 40060) TABLE 8% CASEIN
ET AL.
FORMULATION-
250.0
3.7 438.6 32.0 33.4 150.0 42.3 40.0 10.0
3
TEST DIET (TEKLAD RESEARCH 73065)
FORMULATION-TD
d’Kg Casein, High Protein (87% protein) L-Methionine Sucrose Dextrose, Anhydrous. Technical Dextrin, White. Technical Corn Oil Nonnutritive Fiber (cellulose) Mineral Mix, Rogers-Harper (Cat. No. 170760) Vitamin Mix, Teklad (Cat. No. 40060)
80.0 3.7
580.6 43.0 50.4 150.0 42.3 40.0 10.0
blocks. Analyses included mean daily food intake, water intake and body weight, food and water intakes/l00 g body weight, ratios of food and water intake. incremental growth (added daily body weight) and feeding efficiency (daily weight increment/daily food intake). For each of these dependent measures, a two-way analysis of variance (ageblock X diet) was performed. Separate analyses were performed for males and females. lndiv~dua~ comparisons between diet groups were done by the Newmani~-KLIells procedure [ 151. RESULTS
Although it was considered valuable to include data from rats fed a standard laboratory diet for comparison with data from rats fed either the 25% protein diet or the 8% protein diet, it should be noted that only the two specially prepared diets were isocaloric (4.3 kcal/g). At parturition, the body weights of the neonates did not differ significantly whether from the 8% or 25% dams. However, it was readily apparent in a few days that pups nursing from 8% dams showed reduced growth as defined by body weight. Both males and females weaned from 8%’ diet nurses weighed significantly less than rats weaned from 25% diet nurses (23.8 t 3.0 g vs. 54.8 I 5.0 g for males and 24.7 t 3.5 g vs. 50.0 * 5.8 g for females, means I standard error of the means, both p
Vol. 2, pp. 223-229, 1977. Copyright 0 ANKHO in any form reserved. Printed in the U.S.A.
International
Inc.
Feeding and Drinking in Rats Maintained on a Low Protein Diet’” JOHN E. JALOWIEC3, JANE A. CHISHOLM, WILLIAM B. FORBES, PETER J. MORGANE AND OSCAR RESNICK Worcester Foundation for Ejcperinlental Biology, 222 Maple Avenue, ~~rews~~r~, MA 01545 (Received 22 April 1977) JALOWIEC, J. E., J. A. CHISHOLM, W. B. FORBES, P. J. MORGANE AND 0. RESNICK. Feed&g and dr~nk~lzgin rats ~ajntajned on a low profein diet. BRAIN RES. BULL. Z(3) 223-229, 1977. - Male and female rats born of protein malnourished mothers were fed a low-protein diet (8% casein) for 150 days after weaning and daily food and water intakes and body weights were monitored. Although daily intakes of diet throughout the study were significantly lower than those of rats maintained on a normal protein diet (25% casein) or stock diet, intakes/100 g body weight were significantly greater. Daily increments in body weight, as percent of previous day’s weight, were consistently higher in rats fed the low-protein diet in comparison to rats fed the normal protein diet. Marked retardation of body growth was evident throughout the study although feeding efficiency ratios (daily body wt. increment per g daily food intake) were comparable among the various dietary groups. Compensation for reduction of the protein component of the diet by increased daily food intake/100 g body weight did not alleviate growth retardation. Protein malnutrition Feeding and drinking
Growth behavior
retardation
Incremental
body
weight
Feeding
efficiency
lactation, female rats fed on a low-protein diet did not compensate for reduced protein availability by increasing food intake. The fatter finding is somewhat surprising since the often severe and general debilitation resulting from long-term protein malnutrition could be ameliorated by increased ingestion of the deficient diet. Earlier reports by Adolph [l] and Peterson and Baumgardt [ 10,111 have demonstrated that rats reliably compensate for the addition of non-nutritive bulk to their diets by increasing food intake. Although the importance of dietary protein intake in limiting or increasing total energy intake is unclear, it has been documented that rats can recognize and respond differentially to diets containing various amounts of protein [4]. These reports suggest that rats feeding on diets deficient in protein might adjust their food intake to insure adequate nutrition for growth despite dietary deficiencies. In an attempt to closely paraiiel the human condition, in which mothers are often protein malnourished during gestation and lactation, we initiated protein malnutrition by feeding an 8% protein diet to female rats 5 weeks prior to in~pregnat~on and continued this dietary paradigm throughout gestation and lactation as well as throughout the life of the offspring. In view of studies hy Chow and Lee [ 2)) this paradigm should result in maximal differences between malnourished rats and rats fed a diet containing normal amounts of protein (25%).
IN VIEW OF the inherent difficulties in specifying and controlling the variables associated with protein malnutrition in the human condition f 131, animal models, using rats fed diets deficient in protein, but not calories, have become especially important. Various dietary paradigms have been employed ranging from protein deprivation prior to or at the time of weaning to life-long protein deprivation. Although the generality of findings from experiments using rats to the human condition can be readily assumed on the basis of developmental similarities between species [ 31, generality between findings from different laboratories is often tenuous because of the absence of critical normative data describing the patterns of growth and food intake for rats subjected to different degrees and periods of protein deprivation. For example, a model of malnutrition using protein deprivation only before weaning may not be easily compared to a model based on life-long protein deprivation. In addition, the degree of protein deprivation may be modified by significant differences in food intake between normal and malnourished rats. A recent report suggests that female rats fed a low-protein diet compensate for inadequate protein by ingesting more of the low-protein diet, thereby ingesting more calories and more protein during periods of physiological stress such as pregnancy [7]. However, during periods of mature growth or during
‘Supported by a grant from the National Institute of Health (HD06364). ‘Address correspondence to: Dr. Peter J. Morgane, Worcester Foundation for Experimental Biology, ‘Present address: Department of Psychology, Mount Holyoke College, South Hadley, MA, 223
Shrewsbury.
MA (11545
JALOWIEC
224 The present report describes the alterations in food and water intake and body weight of male and female rats born of mothers maintained on a low-protein diet prior to mating and during gestation and lactation, and themselves maintained on the deficient diet for 150 days after weaning. Data from animals fed a standard laboratory diet as well as from rats fed an isocaloric normal protein diet have been included for comparison. METHOD
Five weeks prior to mating, normal female albino rats (Sprague-Dawley strain, Charles River Breeding Laboratories, Wilmington, MA) were ailowed ad lib access to water and ~onlmercially prepared diets (General Bio~hemi~a~s, Chagrin Falls, OH) containing either 8% or 25% casein as the source of protein. An additional group was allowed access to water and powdered standard laboratory diet (Charles River Rat Formula 4RF). The diet compositions for the 2 test diets and Charles River Rat Formula are given in Tables 1, 2, 3, and 4. The salt mix for each of the test diets is that of Rogers and Harper [ 121. The vitamin mix used for each test diet is given in Table 5. The females were mated with normal males and, after delivery of the young, litter size was reduced to 8 and neonates were randomly redistributed among mothers delivering on the same day and fed the same diet. On Day 21 postpartum, 6 males and 6 females from each dietary regimen were weaned and housed individually (cage size - 45 cm x 27 cm x 27 cm, L., W., D.) with free access to water and the diet of their mother.
1
TABLE
PERCENT NUTRITIONAL COMPOSITION OF
Component Protein Fat Carbohydrate Salt Mix Vitamin Mix Water Nonnutritive Filler Kcal/g
DIET 25%’ Casein Riet*
80/c Casein Diet*
Stock Diet*
21.8 15.4 50.9 4.7 1.0 2.2 4.1 4.3
7.0 15.1 67.9 4.7 1.0 0.9 4.2 4.3
26.3 7.1 42.9 3.5 0.3 9.6 10.3 3.0
*These diets were supplemented casein
lacks this essential
DIETS
amino
with L-methionine
(0.4%)
since
acid.
Beginning on the day of weaning and continuing for 150 days, water intakes from calibrated drinking tubes (k 1 .O ml with steel spouts, food intakes (by weighing spillresistant food containers) and body weights (to the nearest gram) were measured. Food cups and water tubes were refilled every other day. Except for the measurement period (9:00- 1 1 :00 a.m.) the animals were left undisturbed in a temperature-controlled (23”) room with a 12 hr/ I 2 hr light/dark cycle. Analyses of the data were carried out with the aid of a general purpose computer (G.E. time sharing). For the purposes of this report, intakes of water and food and daily body weights were grouped into 15 consecutive IO-day
TABLE 25% CASEIN
TEST
2
DIET (TEKLAD RESEARCH TD 73066)
Casein, High Protein L-~~ethionine Sucrose Dextrose, Anhydrous, Technical Dextrin. White, Technical Corn Oil Nonnutritive Fiber (cellulose) Mineral Mix, Rogers-Harper (Cat. No. 170760) Vitamin Mix, Teklad (Cat. NO. 40060) TABLE 8% CASEIN
ET AL.
FORMULATION-
250.0
3.7 438.6 32.0 33.4 150.0 42.3 40.0 10.0
3
TEST DIET (TEKLAD RESEARCH 73065)
FORMULATION-TD
d’Kg Casein, High Protein (87% protein) L-Methionine Sucrose Dextrose, Anhydrous. Technical Dextrin, White. Technical Corn Oil Nonnutritive Fiber (cellulose) Mineral Mix, Rogers-Harper (Cat. No. 170760) Vitamin Mix, Teklad (Cat. No. 40060)
80.0 3.7
580.6 43.0 50.4 150.0 42.3 40.0 10.0
blocks. Analyses included mean daily food intake, water intake and body weight, food and water intakes/l00 g body weight, ratios of food and water intake. incremental growth (added daily body weight) and feeding efficiency (daily weight increment/daily food intake). For each of these dependent measures, a two-way analysis of variance (ageblock X diet) was performed. Separate analyses were performed for males and females. lndiv~dua~ comparisons between diet groups were done by the Newmani~-KLIells procedure [ 151. RESULTS
Although it was considered valuable to include data from rats fed a standard laboratory diet for comparison with data from rats fed either the 25% protein diet or the 8% protein diet, it should be noted that only the two specially prepared diets were isocaloric (4.3 kcal/g). At parturition, the body weights of the neonates did not differ significantly whether from the 8% or 25% dams. However, it was readily apparent in a few days that pups nursing from 8% dams showed reduced growth as defined by body weight. Both males and females weaned from 8%’ diet nurses weighed significantly less than rats weaned from 25% diet nurses (23.8 t 3.0 g vs. 54.8 I 5.0 g for males and 24.7 t 3.5 g vs. 50.0 * 5.8 g for females, means I standard error of the means, both p
