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Body weights of C3H/HeN mice fed semipurified or commercial diets of different fat content Jerald Silverman
a c
& Jean D. Powers
b
a
Department of Veterinary Preventive Medicine and The Comprehensive Cancer Center , The Ohio State University , Columbus, OH, 43210 b
Departments of Statistics and Veterinary Clinical Sciences , The Ohio State University , Columbus, OH, 43210 c
The Ohio State University , 6089 Godown Rd., Columbus, OH, 43235 Published online: 04 Aug 2009.
To cite this article: Jerald Silverman & Jean D. Powers (1991) Body weights of C3H/HeN mice fed semipurified or commercial diets of different fat content, Nutrition and Cancer, 15:2, 121-127, DOI: 10.1080/01635589109514119 To link to this article: http://dx.doi.org/10.1080/01635589109514119
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms
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Body Weights of C3H/HeN Mice Fed Semipurified or Commercial Diets of Different Fat Content Jerald Silverman and Jean D. Powers
Abstract Oncology studies often require specially formulated diets to be fed to laboratory animals. To determine the effect of dietary fat on body weight, C3H/HeN mice were carefully assigned by weight into three groups. The first group was fed a high-fat semipurified diet (23 % fat by weight) from 21 to 73 days of age then returned to a low-fat semipurified diet (5% fat by weight). A second group was fed the low-fat diet from 21 to 73 days of age, then the high-fat diet until 129 days of age, then returned to the low-fat diet. A final group was fed a 4.5% fat commercial diet for the entire 193-day study. The results showed that the mice fed the semipurified diets did not differ significantly from each other in weight over the course of the study but did differ significantly between 21 and 73 days of age, possibly from a taste preference for the high-fat diet. Mice fed the commercial diet always weighed significantly less. It was estimated that mice fed the commercial diet consumed more food and were less efficient in their food utilization. Mice should be carefully assigned, by weight, into experimental groups, and all groups, including untreated controls, should be fed the same type of diet. (Nutr Cancer 15, 121-127, 1991)
Introduction
For half a century, researchers have studied the effect of the consumption of differing levels of dietary fat on the modulation of carcinogenesis of the mammary gland, colon, and other organs of laboratory rodents. If rodents do not consume properly formulated highand low-fat diets in an isocaloric manner, not only can there be a confounding effect of calories on carcinogenesis, but micronutrients such as vitamin A and selenium (which also can effect carcinogenesis) may also be consumed in differing amounts. In many oncology studies, it was found that rodents had statistically equivalent body J. Silverman is affiliated with the Department of Veterinary Preventive Medicine and The Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210. J.D. Powers is affiliated with the Departments of Statistics and Veterinary Clinical Sciences, The Ohio State University, Columbus, OH 43210.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
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weights when fed diets with different caloric densities resulting from different fat percentages (1-4). However, other researchers found that body weights differed when animals were fed high- or low-fat diets (5-8). In still other work, body weights were statistically different when animals were fed some diets but not others, even when the same fat percentage was present (9). Much of the previous work was performed in rats. In our own studies with mice using high- and low-fat diets, we found in one experiment that body weight differences occurred, whereas in a second more controlled trial, no such differences were seen (4). Therefore, a study was undertaken wherein mice were carefully matched by weight and randomly assigned into experimental groups. We examined the effects on body weight, food consumption, and food use efficiency of changing mice from high-fat to low-fat semipurified diets at different ages. We also studied whether feeding a commercial low-fat mouse diet led to changes in these parameters compared with the semipurified diets. Materials and Methods Animals and Husbandry C3H/HeN virgin female mice (mouse mammary tumor virus negative) were purchased from Charles River Breeding Laboratories (Portage, MI). All animals were certified by the vendor to be viral antibody free and free of significant bacterial and mycoplasmal contaminants. No health examinations were performed during the course of the study. When received at 21 days of age, they were individually weighed, and the mean weights ( ± 2 SD) were included in the study. The 75 animals initially used for the study were divided into high-, medium-, or low-weight groups, each group being approximately one-third of the weight range. Using a table of random numbers, one mouse of each weight group was placed in a cage, and because five mice were kept in each cage, the remaining two were selected from the table of random numbers. Thus, each cage's average weight was approximately the same (±5%), and within each cage the weights of individual mice were approximately the same. Animals were housed in polycarbonate cages (24 x 40 cm). Room temperature was 21 ± 2°C, and relative humidity was 40-60%. There were at least 15 air changes/hour. Hardwood chip bedding was used in all cages, and food and cages were changed twice a week. Water was always available from bottles. Mice were individually weighed once a week. Food consumption was estimated weekly, per cage, by weighing the food available in the food hopper and then reweighing it 24 hours later. Spilled food was not weighed. Diets and Diet Changes Prior to arrival, the mice were fed a commercial rodent diet. After assigning the mice into cages, they were fed one of three different diets (Table 1). Group 1 {n = 25) was fed a high-fat semipurified diet (23% lard) until 73 days of age and was then fed a low-fat semipurified diet (5°7b lard). Group 2 {n = 20 due to accidental loss of 1 cage 3 days into study) was fed the 5% fat diet until 73 days of age, switched to the 23% fat diet until 129 days of age, and then returned to the 5% fat diet. Group 3 (« = 25) was only fed a 4.5% fat commercial diet (LFComm) that had a somewhat lower caloric content than the semipurified 5% fat diet. The changes between 5% and 23% fat diets in Groups 1 and 2 were designed to simulate dietary crossover studies performed in carcinogenesis research. The study continued until the mice were 193 days of age. Diets were prepared in pelleted form, and the 23% fat and 5% fat diets were made by a commercial diet manufacturer (Bioserv, Frenchtown, NJ). The two semipurified diets were
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Table 1. Percentage Composition of Diets" Ingredient
5% Fat Diet, g
23% Fat Diet, g
20.00 0.30 52.00 13.00 5.00 5.00 3.50 1.00 0.20
23.50 0.35 32.90 8.30 5.90 23.52 4.11 1.18 0.24
100.00 3.88
100.00 4.73
Casein, vitamin free DL-Methionine Cornstarch Dextrose Alphacel Lard Mineral mix, AIN Vitamin mix, AIN Choline bitartrate
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Total Metabolic energy, kcal/g
a: Low fat commercial diet (Purina 5001) contains a variety of protein, fat, and carbohydrate sources. Typical composition is protein, 23.4%; fat, 4.5%; crude fiber, 5.8%; nitrogen-free extract, 49.0%; metabolic energy, 3.3 kcal/g. Adequate vitamins and minerals are added.
analyzed for the manufacturer by an independent laboratory for theoretical versus actual nutrient content. There were no significant differences. The LFComm diet (Purina 5001 Rodent Laboratory Chow, Ralston Purina, St. Louis, MO) was not analyzed. All three diets were kept refrigerated, and the 23% fat and 5% fat diets were not used for more than 30 days past manufacturing date. The LFComm fat diet was not used for more than 180 days past the milling date. Statistical Evaluation Because one cage of mice in the 5% fat group died at 24 days of age, the study design was unbalanced, and computer time was restricted, we examined seven time points relative to body weights when the entire study was evaluated. These were 21, 49, 79, 107, 132, 160, and 186 days of age. When statistical differences were found by analysis of variance, StudentNewman-Keuls posttest was used to determine the relationships among the means. During each individual time period (Table 2), body weights were evaluated using five to eight time points (Figure 1). Results Body Weights When the entire course of the study was evaluated, as anticipated, there were no statistically significant body weight differences between Groups 1 and 2, which were fed the semipurified diets (means were 24.6 and 24.1 g, respectively). These two groups did differ _ ,5
2Krol KFot
strut
LFCcmn S2
1CD
>U
IX
Figure 1. Body weights of C3H/HeN mice fed semipurified or commercial diets at different ages.
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Table 2. Body Weight, Estimated Food Consumption, Estimated Caloric Consumption, and Estimated Food Efficiency Mean (SEM)°-*-c
Initial
Body weight, g
Day 21
Day 21-72
Diet
Day 73-128
Diet
Day 129-19:!
Diet
Final Day 193
1
8.6 8.5 8.6
19.7 (2.1)* 16.1 (1.5)* 17.5 (1.7)*
23%Fat 5% Fat LFComm
26.1 (0.4)* 26.2 (1.5)* 23.1 (0.3)*
5% Fat 23% Fat LFComm
30.1 (1.1)* 30.3 (0.5)* 25.8 (0.7)*
5% Fat 5% Fat LFComm
33.1 32.3 27.2
2.4 [11.4] 2.6 [10.1] 3.4 [11.2]
23%Fat 5% Fat LFComm
2.9 [11.3] 2.7 [12.8] 3.5 [11.2]
5% Fat 23% Fat LFComm
3.4 [13.2] 3.2 [12.4] 3.6 [11.9]
5% Fat 5% Fat LFComm
0.13 0.17 0.20
23% Fat 5% Fat LFComm
0.11 0.10 0.15
5% Fat 23% Fat LFComm
0.12 0.10 0.14
5% Fat 5% Fat LFComm
1 2 3
Estimated food efficiency, g consumed/g body wt/day
Mean (SEM)"-*-c
Group 2 3
Estimated food consumption, g/mouse/day
Mean (SEM)"-*-c
1 2 3
a: Values in brackets are expressed in kilocalories. b: Symbols *, *, *: same superscript indicates no significant differences (p > 0.05). c: LFComm, low-fat (4.5% fat) commercial diet •
from mice fed the LFComm diet (mean was 21.8 g, p = 0.005). Over the entire study, all groups exhibited a consistent gain in weight, which was typical for mice of their respective ages. When individual time segments were examined, it was found that differences occurred as shown in Table 2. In general, a change to the 5% fat diet resulted in an immediate decrease in weight, which then slowly increased. When mice were changed to the 23% fat diet, there was a rapid weight gain. Mice fed the LFComm diet consistently had the lowest body weights.
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Food Consumption Estimated food consumption was only evaluated within each of the three time periods, because the dietary changes made in Groups 1 and 2 did not allow for a cumulative evaluation of the entire study period. Table 2 shows that in each of the time periods, mice fed the LFComm diet are estimated to have consumed more food. The estimated caloric consumption (caloric content of diet x average food consumption) of mice in the three groups is also shown in Table 2. Statistical evaluations of food and caloric consumption are not presented, because food wastage was not measured. Food Utilization Efficiency The last parameter we studied was the efficiency of food utilization (food consumption/ gram of body weight/day). Again, comparisons were only made within each of the three time periods of Table 2. In each time period, mice consuming the LFComm diet are estimated to have consumed more food per day for each gram of body weight. A statistical analysis is not presented for the reason indicated previously. Discussion The results of this study show that in female C3H/HeN mice, statistically equivalent body weights resulted from feeding the two closely related semipurified diets, which differed primarily in fat and carbohydrate percentages. However, body weights between mice that were fed the relatively different semipurified 5% fat and LFComm diets differed significantly, even though the fat percentage in each was nearly the same. We cannot definitely state whether the diets were consumed isocalorically, because spillage was not measured, allowing only for estimates of food consumption and food use efficiency. In many research reports, the initial body weights of the animals used were not provided, nor were the details of the randomization of animals into experimental groups. However, Cohen and co-workers (1) were careful to assure equal animal weights in each cage on a per animal basis and reported no significant weight differences during the course of their study between rats fed high- and low-fat semipurified diets. In this study, we carefully assigned mice into experimental groups using essentially the same procedures as Cohen and co-workers (1) and except for the first time period studied (21-73 days of age), we also found no statistically significant differences between the groups fed the two semipurified diets. Other investigators have found statistically significant animal body weight differences and/or differences in food intake in diets that differed in fat content (5-8). In still other studies, rats exhibited significantly different body weights when the same percentage of fat was fed, but the fat came from different sources (coconut oil vs. corn oil) (9). Thus, there are no clear patterns for comparative purposes. Beth and colleagues (10) found that when rats were given diets of different fat content but were restricted to the same caloric consumption, no differences in body weights were seen. This would appear to indicate that in the normal rat, there is not an enhanced utilization of
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dietary fat that is related to the fat content of the diet. However, Wood and Reid (11) fed rats the same number of calories and observed a greater weight gain on a high-fat diet. Earlier studies with mice indicated that the efficiency of food utilization for growth in some mouse strains increased with an increasing fat content of the diet (12). A similar pattern was seen in our studies. In this study, therefore, it might be argued that mice were better able to utilize the semipurified diets for body weight gain compared with the LFComm diet, because the mouse strain, housing conditions, and initial body weights were the same. The estimated food use efficiency data of Table 2 suggest this conclusion. This should not be interpreted as indicating that the semipurified diets were "better" than the LFComm diet, because this study did not address optimal body weights, reproductive performance, longevity, disease resistance, or any other of the many factors that can be influenced by nutrition. Further, we do not presume that dietary fat is of more significance than other nutrients in an animal's ration. A further consideration for the lower weights seen in the LFComm group might be the "filling-up" effect of a diet that is unusually high in fiber. However, no such diet was used in our studies, all diets having approximately 5-6% crude fiber. We might also consider differences in taste preference. Hamilton added 0-30% lard to a commercial pelleted rat diet and concluded that adult male rats had a preference for calorically dense diets (13). Thus, the mice simply might not have liked the LFComm diet as much as the others. This is possible as Figure 1 shows a rapid and fairly continuous weight gain when mice were switched from a low-fat to a high-fat diet and the reverse when switched from a high-fat to low-fat diet. This may also help explain the significantly lower body weights seen in mice consuming the 5% fat diet between days 21 and 73 of age. However, no taste preference trials were performed in our study. Strain and stock differences might be considered factors complicating the comparison of many dietary fat studies in the biomedical literature. As has been noted, a 20% fat diet produces obesity in different strains and stocks of laboratory animals (5,14). Yet, using similar diets, Scholar and others (8) found increased body weights with BALB/c mice, but Fenton and Carr (15) found no such differences with this strain. Kalamegham and Carroll (9) and McCormick (14) found body weight differences with Sprague-Dawley rats, whereas Sylvester and co-workers (2) did not. Similarly, dietary fat-related weight differences may (6) or may not (1) occur with F344 rats. As suggested by this study, careful grouping of experimental animals will lessen the possibility of differences in body weights occurring between experimental groups that are fed similar diets of different fat content. This alone may explain the differences seen in the oncology literature. Additionally, unless experimental needs dictate otherwise, it appears prudent to feed all groups the same general type of diet, including untreated controls, to decrease the possibility of diet-related experimental variability. Acknowledgments and Notes The authors than Dr. Joseph Knapka for his critical review and helpful comments. Address reprint requests to Dr. J. Silverman, The Ohio State University, 6089 Godown Rd., Columbus, OH 43235. Submitted 17 July 1990; accepted in final form 24 October 1990.
References 1. Cohen, L, Thompson, D, Maeura, Y, Choi, K, Blank, M, et al.: "Dietary Fat and Mammary Cancer. 1. Promoting Effects of Different Dietary Fats on Af-Nitrosomethylurea-Induced Rat Mammary Tumorigenesis." JNCI 77, 33-42, 1986. 2. Sylvester, P, Ip, C, and Ip, M: "Effects of High Dietary Fat on the Growth and Development of Ovarian-Independent Carcinogen-Induced Mammary Tumors in Rats." Cancer Res 46, 763-769, 1986.
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3. Gabor, H, and Abraham, S: "Effect of Dietary Menhaden Oil on Tumor Cell Loss and the Accumulation of Mass of a Transplantable Mammary Adenocarcinoma in BALB/c Mice." JNCI 76, 1223-1229, 1986. 4. Silverman, J, Powers, J, Stromberg, P, Pultz, J, and Kent, S: "Effects on C3H Mouse Mammary Cancer of Changing From a High-Fat to a Low-Fat Diet Before, At, or After Puberty." Cancer Res 49, 3857-3860, 1989. 5. Rattigan, S, Howe, P, and Clark, M: "The Effect of a High-Fat Diet and Sucrose Drinking on the Development of Obesity in Spontaneously Hypertensive Rats." Br J Nutr 56, 73-80, 1986. 6. Boissonneault, G, Elson, C, and Pariza, M: "Net Energy Effects of Dietary Fat on Chemically Induced Mammary Carcinogenesis in F344 Rats." JNCI 76, 335-338, 1986. 7. Kraegen, E, Storlien, L, Jenkins, A, and James, D: "Chronic Exercise Compensates for Insulin Resistance Induced by High-Fat Diet in Rats." Am J Physiol 256, E242-E249, 1989. 8. Scholar, E, Violi, L, Newland, J, Bresnick, E, and Birt, D: "The Effect of Dietary Fat on Metastases of the Lewis Lung Carcinoma and the BALB/c Mammary Carcinoma." Nutr Cancer 12, 109-119, 1989. 9. Kalamegham, R, and Carroll, K: "Reversal of the Promotional Effect of High-Fat Diet on Mammary Tumorigenesis By Subsequent Lowering of Dietary Fat." Nutr Cancer 6, 22-31, 1984. 10. Beth, M, Berger, M, Saksoy, M, and Schmahl, D: "Comparison Between the Effects of Dietary Fat Level and of Calorie Intake on Methylnitrosourea-Induced Mammary Carcinogenesis in Female SD Rats." lnt J Cancer 39, 737-744, 1987. 11. Wood, J, and Reid, J: "The Influence of Dietary Fat on Fat Metabolism and Body Fat Deposition in Meal-Feeding and Nibbling Rats." Br J Nutr 34, 15-24, 1975. 12. Hamilton, C: "Rat's Preference for High Fat Diets." J Comp Physio! Psychol 58, 459-460, 1964. 13. Schemmel, R, Michelson, O, and Tolgay, Z: "Dietary Obesity in Rats: Influence of Diet, Weight, Age, and Sex on Body Composition." J Physiol 210, 373-379, 1969. 14. McCormick, D: "Is the Enhancement of Rat Mammary Carcinogenesis by Dietary Fat a Function of Caloric Intake?" Proc Am Assoc Cancer Res 30, 196, 1989. 15. Fenton, P, and Carr, C: "The Nutrition of the Mouse. XI. Responses of Four Strains to Diets Differing in Fat Content." J Nutr 45, 225-233, 1951.
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Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Body weights of C3H/HeN mice fed semipurified or commercial diets of different fat content Jerald Silverman
a c
& Jean D. Powers
b
a
Department of Veterinary Preventive Medicine and The Comprehensive Cancer Center , The Ohio State University , Columbus, OH, 43210 b
Departments of Statistics and Veterinary Clinical Sciences , The Ohio State University , Columbus, OH, 43210 c
The Ohio State University , 6089 Godown Rd., Columbus, OH, 43235 Published online: 04 Aug 2009.
To cite this article: Jerald Silverman & Jean D. Powers (1991) Body weights of C3H/HeN mice fed semipurified or commercial diets of different fat content, Nutrition and Cancer, 15:2, 121-127, DOI: 10.1080/01635589109514119 To link to this article: http://dx.doi.org/10.1080/01635589109514119
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms
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& Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
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Body Weights of C3H/HeN Mice Fed Semipurified or Commercial Diets of Different Fat Content Jerald Silverman and Jean D. Powers
Abstract Oncology studies often require specially formulated diets to be fed to laboratory animals. To determine the effect of dietary fat on body weight, C3H/HeN mice were carefully assigned by weight into three groups. The first group was fed a high-fat semipurified diet (23 % fat by weight) from 21 to 73 days of age then returned to a low-fat semipurified diet (5% fat by weight). A second group was fed the low-fat diet from 21 to 73 days of age, then the high-fat diet until 129 days of age, then returned to the low-fat diet. A final group was fed a 4.5% fat commercial diet for the entire 193-day study. The results showed that the mice fed the semipurified diets did not differ significantly from each other in weight over the course of the study but did differ significantly between 21 and 73 days of age, possibly from a taste preference for the high-fat diet. Mice fed the commercial diet always weighed significantly less. It was estimated that mice fed the commercial diet consumed more food and were less efficient in their food utilization. Mice should be carefully assigned, by weight, into experimental groups, and all groups, including untreated controls, should be fed the same type of diet. (Nutr Cancer 15, 121-127, 1991)
Introduction
For half a century, researchers have studied the effect of the consumption of differing levels of dietary fat on the modulation of carcinogenesis of the mammary gland, colon, and other organs of laboratory rodents. If rodents do not consume properly formulated highand low-fat diets in an isocaloric manner, not only can there be a confounding effect of calories on carcinogenesis, but micronutrients such as vitamin A and selenium (which also can effect carcinogenesis) may also be consumed in differing amounts. In many oncology studies, it was found that rodents had statistically equivalent body J. Silverman is affiliated with the Department of Veterinary Preventive Medicine and The Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210. J.D. Powers is affiliated with the Departments of Statistics and Veterinary Clinical Sciences, The Ohio State University, Columbus, OH 43210.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
Downloaded by [University of Toronto Libraries] at 12:11 30 December 2014
weights when fed diets with different caloric densities resulting from different fat percentages (1-4). However, other researchers found that body weights differed when animals were fed high- or low-fat diets (5-8). In still other work, body weights were statistically different when animals were fed some diets but not others, even when the same fat percentage was present (9). Much of the previous work was performed in rats. In our own studies with mice using high- and low-fat diets, we found in one experiment that body weight differences occurred, whereas in a second more controlled trial, no such differences were seen (4). Therefore, a study was undertaken wherein mice were carefully matched by weight and randomly assigned into experimental groups. We examined the effects on body weight, food consumption, and food use efficiency of changing mice from high-fat to low-fat semipurified diets at different ages. We also studied whether feeding a commercial low-fat mouse diet led to changes in these parameters compared with the semipurified diets. Materials and Methods Animals and Husbandry C3H/HeN virgin female mice (mouse mammary tumor virus negative) were purchased from Charles River Breeding Laboratories (Portage, MI). All animals were certified by the vendor to be viral antibody free and free of significant bacterial and mycoplasmal contaminants. No health examinations were performed during the course of the study. When received at 21 days of age, they were individually weighed, and the mean weights ( ± 2 SD) were included in the study. The 75 animals initially used for the study were divided into high-, medium-, or low-weight groups, each group being approximately one-third of the weight range. Using a table of random numbers, one mouse of each weight group was placed in a cage, and because five mice were kept in each cage, the remaining two were selected from the table of random numbers. Thus, each cage's average weight was approximately the same (±5%), and within each cage the weights of individual mice were approximately the same. Animals were housed in polycarbonate cages (24 x 40 cm). Room temperature was 21 ± 2°C, and relative humidity was 40-60%. There were at least 15 air changes/hour. Hardwood chip bedding was used in all cages, and food and cages were changed twice a week. Water was always available from bottles. Mice were individually weighed once a week. Food consumption was estimated weekly, per cage, by weighing the food available in the food hopper and then reweighing it 24 hours later. Spilled food was not weighed. Diets and Diet Changes Prior to arrival, the mice were fed a commercial rodent diet. After assigning the mice into cages, they were fed one of three different diets (Table 1). Group 1 {n = 25) was fed a high-fat semipurified diet (23% lard) until 73 days of age and was then fed a low-fat semipurified diet (5°7b lard). Group 2 {n = 20 due to accidental loss of 1 cage 3 days into study) was fed the 5% fat diet until 73 days of age, switched to the 23% fat diet until 129 days of age, and then returned to the 5% fat diet. Group 3 (« = 25) was only fed a 4.5% fat commercial diet (LFComm) that had a somewhat lower caloric content than the semipurified 5% fat diet. The changes between 5% and 23% fat diets in Groups 1 and 2 were designed to simulate dietary crossover studies performed in carcinogenesis research. The study continued until the mice were 193 days of age. Diets were prepared in pelleted form, and the 23% fat and 5% fat diets were made by a commercial diet manufacturer (Bioserv, Frenchtown, NJ). The two semipurified diets were
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Table 1. Percentage Composition of Diets" Ingredient
5% Fat Diet, g
23% Fat Diet, g
20.00 0.30 52.00 13.00 5.00 5.00 3.50 1.00 0.20
23.50 0.35 32.90 8.30 5.90 23.52 4.11 1.18 0.24
100.00 3.88
100.00 4.73
Casein, vitamin free DL-Methionine Cornstarch Dextrose Alphacel Lard Mineral mix, AIN Vitamin mix, AIN Choline bitartrate
Downloaded by [University of Toronto Libraries] at 12:11 30 December 2014
Total Metabolic energy, kcal/g
a: Low fat commercial diet (Purina 5001) contains a variety of protein, fat, and carbohydrate sources. Typical composition is protein, 23.4%; fat, 4.5%; crude fiber, 5.8%; nitrogen-free extract, 49.0%; metabolic energy, 3.3 kcal/g. Adequate vitamins and minerals are added.
analyzed for the manufacturer by an independent laboratory for theoretical versus actual nutrient content. There were no significant differences. The LFComm diet (Purina 5001 Rodent Laboratory Chow, Ralston Purina, St. Louis, MO) was not analyzed. All three diets were kept refrigerated, and the 23% fat and 5% fat diets were not used for more than 30 days past manufacturing date. The LFComm fat diet was not used for more than 180 days past the milling date. Statistical Evaluation Because one cage of mice in the 5% fat group died at 24 days of age, the study design was unbalanced, and computer time was restricted, we examined seven time points relative to body weights when the entire study was evaluated. These were 21, 49, 79, 107, 132, 160, and 186 days of age. When statistical differences were found by analysis of variance, StudentNewman-Keuls posttest was used to determine the relationships among the means. During each individual time period (Table 2), body weights were evaluated using five to eight time points (Figure 1). Results Body Weights When the entire course of the study was evaluated, as anticipated, there were no statistically significant body weight differences between Groups 1 and 2, which were fed the semipurified diets (means were 24.6 and 24.1 g, respectively). These two groups did differ _ ,5
2Krol KFot
strut
LFCcmn S2
1CD
>U
IX
Figure 1. Body weights of C3H/HeN mice fed semipurified or commercial diets at different ages.
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Table 2. Body Weight, Estimated Food Consumption, Estimated Caloric Consumption, and Estimated Food Efficiency Mean (SEM)°-*-c
Initial
Body weight, g
Day 21
Day 21-72
Diet
Day 73-128
Diet
Day 129-19:!
Diet
Final Day 193
1
8.6 8.5 8.6
19.7 (2.1)* 16.1 (1.5)* 17.5 (1.7)*
23%Fat 5% Fat LFComm
26.1 (0.4)* 26.2 (1.5)* 23.1 (0.3)*
5% Fat 23% Fat LFComm
30.1 (1.1)* 30.3 (0.5)* 25.8 (0.7)*
5% Fat 5% Fat LFComm
33.1 32.3 27.2
2.4 [11.4] 2.6 [10.1] 3.4 [11.2]
23%Fat 5% Fat LFComm
2.9 [11.3] 2.7 [12.8] 3.5 [11.2]
5% Fat 23% Fat LFComm
3.4 [13.2] 3.2 [12.4] 3.6 [11.9]
5% Fat 5% Fat LFComm
0.13 0.17 0.20
23% Fat 5% Fat LFComm
0.11 0.10 0.15
5% Fat 23% Fat LFComm
0.12 0.10 0.14
5% Fat 5% Fat LFComm
1 2 3
Estimated food efficiency, g consumed/g body wt/day
Mean (SEM)"-*-c
Group 2 3
Estimated food consumption, g/mouse/day
Mean (SEM)"-*-c
1 2 3
a: Values in brackets are expressed in kilocalories. b: Symbols *, *, *: same superscript indicates no significant differences (p > 0.05). c: LFComm, low-fat (4.5% fat) commercial diet •
from mice fed the LFComm diet (mean was 21.8 g, p = 0.005). Over the entire study, all groups exhibited a consistent gain in weight, which was typical for mice of their respective ages. When individual time segments were examined, it was found that differences occurred as shown in Table 2. In general, a change to the 5% fat diet resulted in an immediate decrease in weight, which then slowly increased. When mice were changed to the 23% fat diet, there was a rapid weight gain. Mice fed the LFComm diet consistently had the lowest body weights.
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Food Consumption Estimated food consumption was only evaluated within each of the three time periods, because the dietary changes made in Groups 1 and 2 did not allow for a cumulative evaluation of the entire study period. Table 2 shows that in each of the time periods, mice fed the LFComm diet are estimated to have consumed more food. The estimated caloric consumption (caloric content of diet x average food consumption) of mice in the three groups is also shown in Table 2. Statistical evaluations of food and caloric consumption are not presented, because food wastage was not measured. Food Utilization Efficiency The last parameter we studied was the efficiency of food utilization (food consumption/ gram of body weight/day). Again, comparisons were only made within each of the three time periods of Table 2. In each time period, mice consuming the LFComm diet are estimated to have consumed more food per day for each gram of body weight. A statistical analysis is not presented for the reason indicated previously. Discussion The results of this study show that in female C3H/HeN mice, statistically equivalent body weights resulted from feeding the two closely related semipurified diets, which differed primarily in fat and carbohydrate percentages. However, body weights between mice that were fed the relatively different semipurified 5% fat and LFComm diets differed significantly, even though the fat percentage in each was nearly the same. We cannot definitely state whether the diets were consumed isocalorically, because spillage was not measured, allowing only for estimates of food consumption and food use efficiency. In many research reports, the initial body weights of the animals used were not provided, nor were the details of the randomization of animals into experimental groups. However, Cohen and co-workers (1) were careful to assure equal animal weights in each cage on a per animal basis and reported no significant weight differences during the course of their study between rats fed high- and low-fat semipurified diets. In this study, we carefully assigned mice into experimental groups using essentially the same procedures as Cohen and co-workers (1) and except for the first time period studied (21-73 days of age), we also found no statistically significant differences between the groups fed the two semipurified diets. Other investigators have found statistically significant animal body weight differences and/or differences in food intake in diets that differed in fat content (5-8). In still other studies, rats exhibited significantly different body weights when the same percentage of fat was fed, but the fat came from different sources (coconut oil vs. corn oil) (9). Thus, there are no clear patterns for comparative purposes. Beth and colleagues (10) found that when rats were given diets of different fat content but were restricted to the same caloric consumption, no differences in body weights were seen. This would appear to indicate that in the normal rat, there is not an enhanced utilization of
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dietary fat that is related to the fat content of the diet. However, Wood and Reid (11) fed rats the same number of calories and observed a greater weight gain on a high-fat diet. Earlier studies with mice indicated that the efficiency of food utilization for growth in some mouse strains increased with an increasing fat content of the diet (12). A similar pattern was seen in our studies. In this study, therefore, it might be argued that mice were better able to utilize the semipurified diets for body weight gain compared with the LFComm diet, because the mouse strain, housing conditions, and initial body weights were the same. The estimated food use efficiency data of Table 2 suggest this conclusion. This should not be interpreted as indicating that the semipurified diets were "better" than the LFComm diet, because this study did not address optimal body weights, reproductive performance, longevity, disease resistance, or any other of the many factors that can be influenced by nutrition. Further, we do not presume that dietary fat is of more significance than other nutrients in an animal's ration. A further consideration for the lower weights seen in the LFComm group might be the "filling-up" effect of a diet that is unusually high in fiber. However, no such diet was used in our studies, all diets having approximately 5-6% crude fiber. We might also consider differences in taste preference. Hamilton added 0-30% lard to a commercial pelleted rat diet and concluded that adult male rats had a preference for calorically dense diets (13). Thus, the mice simply might not have liked the LFComm diet as much as the others. This is possible as Figure 1 shows a rapid and fairly continuous weight gain when mice were switched from a low-fat to a high-fat diet and the reverse when switched from a high-fat to low-fat diet. This may also help explain the significantly lower body weights seen in mice consuming the 5% fat diet between days 21 and 73 of age. However, no taste preference trials were performed in our study. Strain and stock differences might be considered factors complicating the comparison of many dietary fat studies in the biomedical literature. As has been noted, a 20% fat diet produces obesity in different strains and stocks of laboratory animals (5,14). Yet, using similar diets, Scholar and others (8) found increased body weights with BALB/c mice, but Fenton and Carr (15) found no such differences with this strain. Kalamegham and Carroll (9) and McCormick (14) found body weight differences with Sprague-Dawley rats, whereas Sylvester and co-workers (2) did not. Similarly, dietary fat-related weight differences may (6) or may not (1) occur with F344 rats. As suggested by this study, careful grouping of experimental animals will lessen the possibility of differences in body weights occurring between experimental groups that are fed similar diets of different fat content. This alone may explain the differences seen in the oncology literature. Additionally, unless experimental needs dictate otherwise, it appears prudent to feed all groups the same general type of diet, including untreated controls, to decrease the possibility of diet-related experimental variability. Acknowledgments and Notes The authors than Dr. Joseph Knapka for his critical review and helpful comments. Address reprint requests to Dr. J. Silverman, The Ohio State University, 6089 Godown Rd., Columbus, OH 43235. Submitted 17 July 1990; accepted in final form 24 October 1990.
References 1. Cohen, L, Thompson, D, Maeura, Y, Choi, K, Blank, M, et al.: "Dietary Fat and Mammary Cancer. 1. Promoting Effects of Different Dietary Fats on Af-Nitrosomethylurea-Induced Rat Mammary Tumorigenesis." JNCI 77, 33-42, 1986. 2. Sylvester, P, Ip, C, and Ip, M: "Effects of High Dietary Fat on the Growth and Development of Ovarian-Independent Carcinogen-Induced Mammary Tumors in Rats." Cancer Res 46, 763-769, 1986.
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3. Gabor, H, and Abraham, S: "Effect of Dietary Menhaden Oil on Tumor Cell Loss and the Accumulation of Mass of a Transplantable Mammary Adenocarcinoma in BALB/c Mice." JNCI 76, 1223-1229, 1986. 4. Silverman, J, Powers, J, Stromberg, P, Pultz, J, and Kent, S: "Effects on C3H Mouse Mammary Cancer of Changing From a High-Fat to a Low-Fat Diet Before, At, or After Puberty." Cancer Res 49, 3857-3860, 1989. 5. Rattigan, S, Howe, P, and Clark, M: "The Effect of a High-Fat Diet and Sucrose Drinking on the Development of Obesity in Spontaneously Hypertensive Rats." Br J Nutr 56, 73-80, 1986. 6. Boissonneault, G, Elson, C, and Pariza, M: "Net Energy Effects of Dietary Fat on Chemically Induced Mammary Carcinogenesis in F344 Rats." JNCI 76, 335-338, 1986. 7. Kraegen, E, Storlien, L, Jenkins, A, and James, D: "Chronic Exercise Compensates for Insulin Resistance Induced by High-Fat Diet in Rats." Am J Physiol 256, E242-E249, 1989. 8. Scholar, E, Violi, L, Newland, J, Bresnick, E, and Birt, D: "The Effect of Dietary Fat on Metastases of the Lewis Lung Carcinoma and the BALB/c Mammary Carcinoma." Nutr Cancer 12, 109-119, 1989. 9. Kalamegham, R, and Carroll, K: "Reversal of the Promotional Effect of High-Fat Diet on Mammary Tumorigenesis By Subsequent Lowering of Dietary Fat." Nutr Cancer 6, 22-31, 1984. 10. Beth, M, Berger, M, Saksoy, M, and Schmahl, D: "Comparison Between the Effects of Dietary Fat Level and of Calorie Intake on Methylnitrosourea-Induced Mammary Carcinogenesis in Female SD Rats." lnt J Cancer 39, 737-744, 1987. 11. Wood, J, and Reid, J: "The Influence of Dietary Fat on Fat Metabolism and Body Fat Deposition in Meal-Feeding and Nibbling Rats." Br J Nutr 34, 15-24, 1975. 12. Hamilton, C: "Rat's Preference for High Fat Diets." J Comp Physio! Psychol 58, 459-460, 1964. 13. Schemmel, R, Michelson, O, and Tolgay, Z: "Dietary Obesity in Rats: Influence of Diet, Weight, Age, and Sex on Body Composition." J Physiol 210, 373-379, 1969. 14. McCormick, D: "Is the Enhancement of Rat Mammary Carcinogenesis by Dietary Fat a Function of Caloric Intake?" Proc Am Assoc Cancer Res 30, 196, 1989. 15. Fenton, P, and Carr, C: "The Nutrition of the Mouse. XI. Responses of Four Strains to Diets Differing in Fat Content." J Nutr 45, 225-233, 1951.
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