- Email: [email protected]
Operant Responding for Dietary Protein in the Golden Hamster (Mesocricetus auratus)
Physiology & Behavior, Vol. 67, No. 1, pp. 95–98, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99/$–see front matter
PII S0031-9384(99)00043-8
Operant Responding for Dietary Protein in the Golden Hamster (Mesocricetus auratus) DAVID DIBATTISTA1 Brock University, Department of Psychology, St. Catharines, Ontario, Canada L2S 3A1 Received 22 September 1998; Accepted 23 February 1999 DiBATTISTA, D. Operant responding for dietary protein in the golden hamster (Mesocricetus auratus). PHYSIOL BEHAV 67(1) 95–98, 1999.—Golden hamsters housed in operant chambers over a period of weeks had ad lib access to a maintenance diet that was either nutritionally complete (NCMD) or protein-free (PFMD), and they were required to press a lever on a fixed-ratio (FR) schedule to obtain 20-mg high-protein pellets. As the FR requirement increased, hamsters maintained on the NCMD made fewer lever presses and ate fewer pellets, and at the highest FR levels, they earned very few pellets. In contrast, hamsters maintained on PFMD increased the number of lever presses as the FR requirement increased, and they only slightly reduced the number of pellets eaten. Even at the highest FR requirement levels, PFMD hamsters still derived an average of 11–12% of total calories from protein, a level of intake that is either adequate for adult hamsters, or very nearly so. Previous research has shown that hamsters make adaptive behavioural adjustments in response to time-restricted access to dietary protein, and the present findings demonstrate that protein-restricted hamsters that must press a lever to obtain protein-rich pellets also make adaptive behavioural adjustments when challenged with increases in the FR requirement. ©1999 Elsevier Science Inc. Golden hamster
Diet selection
Protein selection
Operant responding for protein
It has been shown that rats that are maintained on a protein-free diet and required to press a lever to obtain proteinrich pellets increase their rate of lever pressing as the fixed-ratio requirement is raised over a period of days (2). Because hamsters make adaptive behavioural adjustments in response to time-restricted access to dietary protein, it is reasonable to ask if they will also successfully adapt to changes in the amount of effort that they must expend to obtain dietary protein. An operant conditioning paradigm was employed to investigate this question. Briefly, two groups of hamsters were housed individually in operant conditioning chambers for a period of weeks and had continuous free access to a maintenance diet. For one group, the maintenance diet was nutritionally complete, and for the other group the maintenance diet was isocaloric, but it contained no protein. All hamsters were able to obtain high-protein pellets by pressing a lever on a fixed-ratio schedule, and the effects of gradual increases in the fixed-ratio requirement were observed.
THE ingestive behaviour of golden hamsters is characterized by a variety of striking peculiarities (4,8,13–16). However, hamsters are similar to rats in making adaptive behavioural adjustments in response to time-restricted access to dietary protein (5,6,9–11). For example, when hamsters are maintained on a fractionated diet over a period of days and allowed access to the protein-rich diet component for only 1 h/ day, they substantially increase their consumption of protein as the restriction period continues (5). Furthermore, hamsters’ increased consumption of a protein-rich diet is a highly selective behavioural response. Thus, in a recent study hamsters were maintained for 10 days on either a protein-free or a nutritionally complete maintenance diet, and they were also given access to protein-rich and carbohydrate-rich test diets for 6 h/day (9). Hamsters on the protein-free maintenance diet demonstrated a large and selective increase in their consumption of the protein-rich test diet. In contrast, hamsters on the complete maintenance diet ate equal amounts of the two test diets even when their intake of maintenance diet was matched to that of hamsters on the protein-free maintenance diet. It has also been found that protein-restricted hamsters learn to discriminate between the intermittently presented test diets by associating the orosensory properties of the test diets (i.e., their flavours) with the consequences of ingestion (11). 1To
Food intake
MATERIALS AND METHODS
Subjects and Housing Twelve adult male golden hamsters (Mesocricetus auratus) were purchased from Charles River Canada and weighed 96–
whom requests for reprints should be addressed. E-mail:[email protected]
95
96
DIBATTISTA
128 g at the start of the experiment. Prior to the experiment, hamsters were housed in individual hanging wire cages (25 3 18 3 15 cm) and maintained on pelleted Purina Rodent Chow (No. 5001). The experimental room was kept at 21–238C and illuminated from 0800 to 2200 h. Materials Each 20-mg high-protein pellet (BioServ, Frenchtown, NJ) provided 0.073 kcal and contained 63.9% soy protein, 14.6% cornstarch, 4.3% corn oil, 5.0% vitamins and minerals, 7.9% cellulose, and 6.9% moisture. Composition of the nutritionally complete (NCMD) and protein-free (PFMD) maintenance diets is provided in Table 1. Maintenance diets, both of which provided 4.83 kcal/g, were presented in containers made by gluing a glass jar with a 125-mL capacity inside a larger glass cup. Food was placed in the glass jar with spillage being caught in the surrounding cup. A flat piece of aluminum (12 3 12 3 0.08 cm) with rounded corners was glued to the bottom of the cup to prevent tipping of the food container. Spillage that occurred despite these precautions was rare. Operant conditioning chambers measured 27 3 27 3 24 cm (length 3 width 3 height). Each was equipped with a lever in the middle of the front wall, just to the left of a food trough into which 20-mg food pellets were dispensed. Tap water was available at all times in a bottle on the opposite wall of the chamber. The chamber floor consisted of a series of parallel aluminum rods, 6 mm in diameter and spaced 8 mm apart. Tissue paper was provided to permit hamsters to build nests. Procedure Hamsters were randomly divided into two treatment groups (n 5 6/group) that had ad lib access either to NCMD or to PFMD throughout the experiment. Before the experiment began, hamsters were acclimatised to the operant chambers and trained to lever press to obtain standard 20-mg food pellets (BioServ Rodent Pellets). On the first day of the experiment, Purina Chow was removed from the home cage and hamsters were switched to their respective maintenance diets. Hamsters were also given ad lib access to a second food jar containing high-protein pellets. After 7 days in the home cage under this regimen, hamsters were moved into individual operant conditioning chambers for the remainder of the study. Maintenance diets continued to be available ad lib, but hamsters could now obtain high-protein pellets only by pressing the lever. For the first 2 weeks that hamsters were housed in
the operant chambers, pellets were available on an FR1 schedule. Over the next 5 weeks, the fixed-ratio requirement was gradually raised to FR5, with animals staying at each FR requirement level for 7–10 days. For each hamster, data were averaged over the last 4 days at each FR requirement level. Occasionally, it was possible to obtain data for only 3 days, as the result of equipment malfunction. Each chamber was serviced daily for 15–20 min between 0900 and 1400 h. Each subject was weighed, its water was replenished, and the chamber and the bedding below it were searched for uneaten pellets, which were counted and discarded. Hamsters were moved into clean chambers every 5–7 days. Statistical Analyses Statistical analyses were carried out using the SPSS 8.0 for Windows statistical package. Consumption data were analyzed using a mixed-design analysis of variance (ANOVA) with one between-subjects variable (maintenance diet) and one within-subjects variable (ratio requirement). Corrections for degrees of freedom for within-subjects variables were carried out following the recommendations of Keppel (12). Planned comparisons were carried out using the modified Bonferroni test with alpha set at 0.05 (12). RESULTS
Figure 1 shows the number of lever presses made by hamsters as a function of the fixed-ratio requirement. Overall, PFMD hamsters made more than twice as many lever presses as did NCMD hamsters, F(1, 10) 5 10.3, p , 0.01 (PFMD: mean 6 SE 5 276.9 6 38.6 presses/day; NCMD: 120.5 6 29.5). A significant maintenance diet 3 ratio requirement interaction, F(4, 40) 5 4.20, p , 0.01, indicated that NCMD hamsters pressed less as the FR requirement increased, whereas PFMD hamsters tended to press more. At FR4 and FR5, PFMD hamsters made significantly more lever presses than did NCMD hamsters. Overall, PFMD hamsters earned about twice as many pellets as did NCMD hamsters, F(1, 10) 5 5.01, p , 0.05 (PFMD: 118.6 6 17.8 pellets/day; NCMD: 62.4 6 17.7). However, Fig. 2 indicates that the number of pellets earned by both NCMD and PFMD hamsters fell as the FR requirement increased, F(2, 23) 5 18.5, p , 0.001. At FR 4 and FR5, PFMD hamsters
TABLE 1 COMPOSITION OF MAINTENANCE DIETS AS PERCENTAGE OF TOTAL WEIGHT
Casein* Methionine* Vegetable oil† Cornstarch* Vitamin mixture‡ Mineral mixture§ Alphacel*
Protein-Free Diet
Nutritionally Complete Diet
— — 20 74 1.5 3.5 1
24.5 0.5 20 49 1.5 3.5 1
*ICN Canada. †Crisco oil, Procter and Gamble. ‡AIN Vitamin Mixture 76, ICN Canada. Contains 97.3% sucrose §AIN Mineral Mixture 76, ICN Canada. Contains 11.8% sucrose.
FIG. 1. Mean number of lever presses by hamsters kept on maintenance diets that were either nutritionally complete (NCMD) or protein free (PFMD). Standard errors ranged from 14.1 to 68.4. wSignificantly greater than the corresponding value for the NCMD group.
DIETARY PROTEIN
97
FIG. 3. Mean number of pellets eaten by hamsters kept on maintenance diets that were either nutritionally complete (NCMD) or protein free (PFMD). Standard errors ranged from 1.98 to 15.04. wSignificantly greater than the corresponding value for the NCMD group.
FIG. 2. Mean number of pellets earned and pellets left uneaten by hamsters kept on maintenance diets that were either nutritionally complete (upper panel) or protein free (lower panel). Standard errors ranged from 4.37 to 57.60 for pellets earned and from 0 to 50.4 for pellets left uneaten. wSignificantly greater than the corresponding value for the NCMD group.
continued to earn significantly more pellets than NCMD hamsters, which earned very few pellets (,20 pellets/day). Hamsters on the two maintenance diets did not differ in the total amount of pellets left uneaten, F(1, 10) 5 0.33, NS (PFMD: 34.4 6 13.6 pellets/day; NCMD: 21.5 6 17.5). However, Fig. 2 shows that for both groups, the number of uneaten pellets decreased as the FR requirement increased, F(2, 17) 5 7.21, p , 0.01, with PFMD hamsters leaving no pellets uneaten at both FR4 and FR5. Because the nature of the flooring in the operant chambers did not permit hamsters to hoard pellets effectively, most uneaten pellets were retrieved from the bedding beneath the cage. Figure 3 shows the number of high-protein pellets that were actually eaten by hamsters during the various phases of the study. Overall, PFMD hamsters ate significantly more pellets than did NCMD hamsters, F(1, 10) 5 22.2, p , 0.001 (PFMD: 87.3 6 6.11 pellets/day; NCMD: 49.0 6 5.38). Furthermore, a significant maintenance diet 3 ratio requirement interaction, F(5, 50) 5 4.20, p , 0.05, reflects the fact that increases in the FR requirement had dramatically different effects on pellet consumption for hamsters on the two maintenance diets. When housed in their home cages during the initial phase of the study with high-protein pellets freely available, hamsters on the two maintenance diets ate similar numbers of pellets. However, as the fixed ratio requirement increased, NCMD hamsters demonstrated a dramatic decrease in their pellet consumption, and by the end of the experiment, they were consuming only about 10% as many pellets as they had at the start. In contrast, as the fixed ratio requirement increased, PFMD hamsters only slightly decreased the number of high-protein pellets that they consumed, and they ate significantly more high-protein pellets than did NCMD hamsters at FR3, FR4, and FR5.
Over the course of the experiment, PFMD hamsters derived about 12% less energy from the maintenance diet than did NCMD hamsters, F(1, 10) 5 11.3, p , 0.01 (PFMD: 27.2 6 0.73 kcal/day; NCMD: 30.6 6 0.70). However, the increased consumption of high-protein pellets by PFMD hamsters resulted in there being no significant difference in total energy consumption between the two treatment groups, F(1, 10) 5 0.20, NS (PFMD: 33.5 6 1.02 kcal/day; NCMD: 34.1 6 0.92). Of course, over the course of the experiment PFMD hamsters derived a significantly smaller percentage of total calories from protein than did NCMD hamsters, F(1, 10) 5 233.7, p , 0.001 (PFMD: 13.3 6 0.65%; NCMD: 25.8 6 0.49). Furthermore, although the percentage of protein-derived calories decreased as the FR requirement increased, PFMD hamsters still derived an average of 11–12% of total calories from protein even at the highest FR requirement levels, a level of total protein intake that is either adequate for adult hamsters or very nearly so (1,3). Nevertheless, PFMD hamsters gained significantly less weight than NCMD hamsters over the course of the experiment, t(10) 5 2.31, p , 0.05 (PFMD: 2.8 6 4.1 g; NCMD: 13.8 6 2.5). DISCUSSION
Previous research has shown that hamsters respond adaptively when environmental circumstances make it more difficult than usual for them to obtain sufficient amounts of dietary protein. Specifically, time-restricted access to dietary protein causes hamsters to develop a strong and selective preference for a protein-rich diet that is only intermittently available (5,9,11). A similar pattern of behaviour has also been observed in rats (6,10). The present experiment demonstrates that protein-restricted hamsters that must press a lever to obtain dietary protein also make adaptive behavioural adjustments when they are challenged with increases in the FR requirement. Hamsters maintained on a nutritionally complete diet were found to reduce both their rate of lever pressing and their consumption of high-protein pellets when the FR requirement was increased, and at the highest FR requirement levels, they earned and ate very few pellets. In contrast, hamsters maintained on a protein-free diet increased their rate of lever pressing when the FR requirement was increased. Moreover, even at the highest FR requirement levels, they achieved an adequate level of dietary protein intake,
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DIBATTISTA
although their weight gain was substantially less than that of hamsters maintained on the nutritionally complete diet, presumably as a result of their reduced protein intake. It is noteworthy that hamsters maintained on the nutritionally complete and the protein-free diets consumed similar amounts of high-protein pellets when the pellets were either freely available or when the FR requirement was low. Behavioural differences between hamsters eating the two different maintenance diets became evident only when the FR requirement increased. Thus, the use of the operant conditioning paradigm permitted the detection of substantial motivational differences between hamsters maintained on the nutritionally complete and the protein-free diets. Mammals make use of protein to fulfill a variety of vital functions and to maintain the structural integrity of the body. However, mammals continuously lose protein from the body in the course of everyday metabolism, and they do not possess specific cells or tissues that are distinctly specialized for the task of storing either amino acids or protein. Therefore, protein must be obtained in the diet on a regular basis for the maintenance of good health. Consequently, although the ingestive behaviour of hamsters is characterized by a variety of striking peculiarities (7,8,13–16), it is not surprising to find that hamsters nevertheless resemble rats in responding adaptively both to time-restricted access to dietary protein (6,10) and as shown here, to increases in the workload required to obtain dietary protein in an operant situation (2). Although the design of the present experiment does not allow one to conclude that protein-restricted hamsters maintained their lever pressing because the pellets were protein rich, the findings
of other studies strongly suggest that this is so. Thus, in a recent experiment, hamsters housed in standard cages were at different times maintained on protein-free and nutritionally complete maintenance diets, and they were also allowed intermittent access to protein-rich and carbohydrate-rich test diets that contained distinctive marker flavours. When later allowed to select between the flavoured test diets in a brief preference test, hamsters strongly preferred the protein-rich test diet when they were being maintained on the protein-free diet, but not when they were being maintained on the nutritionally complete diet. Furthermore, when allowed to select between two nutritionally identical diets that differed only in their marker flavour, hamsters showed a strong preference for the flavour previously associated with the protein-rich test diet when they were being maintained on the protein-free diet, but not when they were being maintained on the nutritionally complete diet (11). In light of these findings, it appears likely that the protein-restricted hamsters in the present experiment learned to associate the orosensory properties of the high-protein pellets with the consequences of their ingestion, and that they adjusted their lever-pressing behaviour in order to maintain an adequate level of dietary protein intake as the FR requirement was increased. ACKNOWLEDGEMENTS
This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to David DiBattista. The author is grateful to Dayle Belme, Rosa Brew, Debbie Chindemi, and Traci DiBattista for their technical assistance.
REFERENCES 1. Arrington, L. R.; Platt, J. K.; Shirley, R. L.: Protein requirements of golden hamsters. Lab. Anim. Care 16:492–496; 1966. 2. Ashley, D. V. M.: Factors affecting the selection of protein and carbohydrate from a dietary choice. Nutr. Res. 5:555–571; 1985. 3. Banta, C. A.; Warner, R. G.; Robertson, J. B.: Protein nutrition of the golden hamster. J. Nutr. 105:38–45; 1975. 4. DiBattista, D.: Effects of 5-thioglucose on feeding and glycemia in the hamster. Physiol. Behav. 29:803–806; 1982. 5. DiBattista, D.: Control of protein intake in golden hamsters. Physiol. Behav. 39:1–10; 1987. 6. DiBattista, D.: Effects of time-restricted access to protein and to carbohydrate in adult mice and rats. Physiol.Behav. 49:263–269; 1991. 7. DiBattista, D.: Voluntary lactose ingestion in gerbils, rats, mice, and golden hamsters. Physiol. Behav. 52:59–63; 1992. 8. DiBattista, D.: Familiarity and flavour preference in the golden hamster (Mesocricetus auratus). (submitted) 9. DiBattista, D.; Campbell, D. A.: Dietary protein restriction and selective preference for a protein-containing diet in the golden hamster (Mesocricetus auratus). Physiol. Behav. 64:563–571; 1998.
10. DiBattista, D.; Holder, M. D.: Enhanced preference for a protein-containing diet in response to dietary protein restriction. Appetite 30:237–254; 1998. 11. DiBattista, D.; Mercier, S. A.: Role of learning in the selection of dietary protein in the golden hamster (Mesocricetus auratus). Behav. Neurosci. (in press). 12. Keppel, G.: Design and analysis: A researcher’s handbook. Englewood Cliffs, NJ: Prentice Hall; 1991. 13. Ksir, C.: Taste and nicotine as determinants of voluntary tobacco use by hamsters. Pharmacol. Biochem. Behav. 19:605–608; 1983. 14. Lazzarini, S. J.; Schneider, J. E.; Wade, G. N.: Effects of fatty acid oxidation and glucose metabolism on food intake and hunger motivation in Syrian hamsters. Physiol. Behav. 44:209–215; 1988. 15. McMillan, D. E.; Ellis, F. W.; Frye, G. D.; Pick, J. R.: Failure of signs of physical dependence to develop in hamsters after prolonged consumption of large doses of ethanol. Pharmacol. Biochem. Behav. 7:55–57; 1977. 16. Silverman, H. J.; Zucker, I.: Absence of post-fast food compensation in the golden hamster (Mesocricetus auratus). Physiol. Behav. 17:271–285; 1976.
PII S0031-9384(99)00043-8
Operant Responding for Dietary Protein in the Golden Hamster (Mesocricetus auratus) DAVID DIBATTISTA1 Brock University, Department of Psychology, St. Catharines, Ontario, Canada L2S 3A1 Received 22 September 1998; Accepted 23 February 1999 DiBATTISTA, D. Operant responding for dietary protein in the golden hamster (Mesocricetus auratus). PHYSIOL BEHAV 67(1) 95–98, 1999.—Golden hamsters housed in operant chambers over a period of weeks had ad lib access to a maintenance diet that was either nutritionally complete (NCMD) or protein-free (PFMD), and they were required to press a lever on a fixed-ratio (FR) schedule to obtain 20-mg high-protein pellets. As the FR requirement increased, hamsters maintained on the NCMD made fewer lever presses and ate fewer pellets, and at the highest FR levels, they earned very few pellets. In contrast, hamsters maintained on PFMD increased the number of lever presses as the FR requirement increased, and they only slightly reduced the number of pellets eaten. Even at the highest FR requirement levels, PFMD hamsters still derived an average of 11–12% of total calories from protein, a level of intake that is either adequate for adult hamsters, or very nearly so. Previous research has shown that hamsters make adaptive behavioural adjustments in response to time-restricted access to dietary protein, and the present findings demonstrate that protein-restricted hamsters that must press a lever to obtain protein-rich pellets also make adaptive behavioural adjustments when challenged with increases in the FR requirement. ©1999 Elsevier Science Inc. Golden hamster
Diet selection
Protein selection
Operant responding for protein
It has been shown that rats that are maintained on a protein-free diet and required to press a lever to obtain proteinrich pellets increase their rate of lever pressing as the fixed-ratio requirement is raised over a period of days (2). Because hamsters make adaptive behavioural adjustments in response to time-restricted access to dietary protein, it is reasonable to ask if they will also successfully adapt to changes in the amount of effort that they must expend to obtain dietary protein. An operant conditioning paradigm was employed to investigate this question. Briefly, two groups of hamsters were housed individually in operant conditioning chambers for a period of weeks and had continuous free access to a maintenance diet. For one group, the maintenance diet was nutritionally complete, and for the other group the maintenance diet was isocaloric, but it contained no protein. All hamsters were able to obtain high-protein pellets by pressing a lever on a fixed-ratio schedule, and the effects of gradual increases in the fixed-ratio requirement were observed.
THE ingestive behaviour of golden hamsters is characterized by a variety of striking peculiarities (4,8,13–16). However, hamsters are similar to rats in making adaptive behavioural adjustments in response to time-restricted access to dietary protein (5,6,9–11). For example, when hamsters are maintained on a fractionated diet over a period of days and allowed access to the protein-rich diet component for only 1 h/ day, they substantially increase their consumption of protein as the restriction period continues (5). Furthermore, hamsters’ increased consumption of a protein-rich diet is a highly selective behavioural response. Thus, in a recent study hamsters were maintained for 10 days on either a protein-free or a nutritionally complete maintenance diet, and they were also given access to protein-rich and carbohydrate-rich test diets for 6 h/day (9). Hamsters on the protein-free maintenance diet demonstrated a large and selective increase in their consumption of the protein-rich test diet. In contrast, hamsters on the complete maintenance diet ate equal amounts of the two test diets even when their intake of maintenance diet was matched to that of hamsters on the protein-free maintenance diet. It has also been found that protein-restricted hamsters learn to discriminate between the intermittently presented test diets by associating the orosensory properties of the test diets (i.e., their flavours) with the consequences of ingestion (11). 1To
Food intake
MATERIALS AND METHODS
Subjects and Housing Twelve adult male golden hamsters (Mesocricetus auratus) were purchased from Charles River Canada and weighed 96–
whom requests for reprints should be addressed. E-mail:[email protected]
95
96
DIBATTISTA
128 g at the start of the experiment. Prior to the experiment, hamsters were housed in individual hanging wire cages (25 3 18 3 15 cm) and maintained on pelleted Purina Rodent Chow (No. 5001). The experimental room was kept at 21–238C and illuminated from 0800 to 2200 h. Materials Each 20-mg high-protein pellet (BioServ, Frenchtown, NJ) provided 0.073 kcal and contained 63.9% soy protein, 14.6% cornstarch, 4.3% corn oil, 5.0% vitamins and minerals, 7.9% cellulose, and 6.9% moisture. Composition of the nutritionally complete (NCMD) and protein-free (PFMD) maintenance diets is provided in Table 1. Maintenance diets, both of which provided 4.83 kcal/g, were presented in containers made by gluing a glass jar with a 125-mL capacity inside a larger glass cup. Food was placed in the glass jar with spillage being caught in the surrounding cup. A flat piece of aluminum (12 3 12 3 0.08 cm) with rounded corners was glued to the bottom of the cup to prevent tipping of the food container. Spillage that occurred despite these precautions was rare. Operant conditioning chambers measured 27 3 27 3 24 cm (length 3 width 3 height). Each was equipped with a lever in the middle of the front wall, just to the left of a food trough into which 20-mg food pellets were dispensed. Tap water was available at all times in a bottle on the opposite wall of the chamber. The chamber floor consisted of a series of parallel aluminum rods, 6 mm in diameter and spaced 8 mm apart. Tissue paper was provided to permit hamsters to build nests. Procedure Hamsters were randomly divided into two treatment groups (n 5 6/group) that had ad lib access either to NCMD or to PFMD throughout the experiment. Before the experiment began, hamsters were acclimatised to the operant chambers and trained to lever press to obtain standard 20-mg food pellets (BioServ Rodent Pellets). On the first day of the experiment, Purina Chow was removed from the home cage and hamsters were switched to their respective maintenance diets. Hamsters were also given ad lib access to a second food jar containing high-protein pellets. After 7 days in the home cage under this regimen, hamsters were moved into individual operant conditioning chambers for the remainder of the study. Maintenance diets continued to be available ad lib, but hamsters could now obtain high-protein pellets only by pressing the lever. For the first 2 weeks that hamsters were housed in
the operant chambers, pellets were available on an FR1 schedule. Over the next 5 weeks, the fixed-ratio requirement was gradually raised to FR5, with animals staying at each FR requirement level for 7–10 days. For each hamster, data were averaged over the last 4 days at each FR requirement level. Occasionally, it was possible to obtain data for only 3 days, as the result of equipment malfunction. Each chamber was serviced daily for 15–20 min between 0900 and 1400 h. Each subject was weighed, its water was replenished, and the chamber and the bedding below it were searched for uneaten pellets, which were counted and discarded. Hamsters were moved into clean chambers every 5–7 days. Statistical Analyses Statistical analyses were carried out using the SPSS 8.0 for Windows statistical package. Consumption data were analyzed using a mixed-design analysis of variance (ANOVA) with one between-subjects variable (maintenance diet) and one within-subjects variable (ratio requirement). Corrections for degrees of freedom for within-subjects variables were carried out following the recommendations of Keppel (12). Planned comparisons were carried out using the modified Bonferroni test with alpha set at 0.05 (12). RESULTS
Figure 1 shows the number of lever presses made by hamsters as a function of the fixed-ratio requirement. Overall, PFMD hamsters made more than twice as many lever presses as did NCMD hamsters, F(1, 10) 5 10.3, p , 0.01 (PFMD: mean 6 SE 5 276.9 6 38.6 presses/day; NCMD: 120.5 6 29.5). A significant maintenance diet 3 ratio requirement interaction, F(4, 40) 5 4.20, p , 0.01, indicated that NCMD hamsters pressed less as the FR requirement increased, whereas PFMD hamsters tended to press more. At FR4 and FR5, PFMD hamsters made significantly more lever presses than did NCMD hamsters. Overall, PFMD hamsters earned about twice as many pellets as did NCMD hamsters, F(1, 10) 5 5.01, p , 0.05 (PFMD: 118.6 6 17.8 pellets/day; NCMD: 62.4 6 17.7). However, Fig. 2 indicates that the number of pellets earned by both NCMD and PFMD hamsters fell as the FR requirement increased, F(2, 23) 5 18.5, p , 0.001. At FR 4 and FR5, PFMD hamsters
TABLE 1 COMPOSITION OF MAINTENANCE DIETS AS PERCENTAGE OF TOTAL WEIGHT
Casein* Methionine* Vegetable oil† Cornstarch* Vitamin mixture‡ Mineral mixture§ Alphacel*
Protein-Free Diet
Nutritionally Complete Diet
— — 20 74 1.5 3.5 1
24.5 0.5 20 49 1.5 3.5 1
*ICN Canada. †Crisco oil, Procter and Gamble. ‡AIN Vitamin Mixture 76, ICN Canada. Contains 97.3% sucrose §AIN Mineral Mixture 76, ICN Canada. Contains 11.8% sucrose.
FIG. 1. Mean number of lever presses by hamsters kept on maintenance diets that were either nutritionally complete (NCMD) or protein free (PFMD). Standard errors ranged from 14.1 to 68.4. wSignificantly greater than the corresponding value for the NCMD group.
DIETARY PROTEIN
97
FIG. 3. Mean number of pellets eaten by hamsters kept on maintenance diets that were either nutritionally complete (NCMD) or protein free (PFMD). Standard errors ranged from 1.98 to 15.04. wSignificantly greater than the corresponding value for the NCMD group.
FIG. 2. Mean number of pellets earned and pellets left uneaten by hamsters kept on maintenance diets that were either nutritionally complete (upper panel) or protein free (lower panel). Standard errors ranged from 4.37 to 57.60 for pellets earned and from 0 to 50.4 for pellets left uneaten. wSignificantly greater than the corresponding value for the NCMD group.
continued to earn significantly more pellets than NCMD hamsters, which earned very few pellets (,20 pellets/day). Hamsters on the two maintenance diets did not differ in the total amount of pellets left uneaten, F(1, 10) 5 0.33, NS (PFMD: 34.4 6 13.6 pellets/day; NCMD: 21.5 6 17.5). However, Fig. 2 shows that for both groups, the number of uneaten pellets decreased as the FR requirement increased, F(2, 17) 5 7.21, p , 0.01, with PFMD hamsters leaving no pellets uneaten at both FR4 and FR5. Because the nature of the flooring in the operant chambers did not permit hamsters to hoard pellets effectively, most uneaten pellets were retrieved from the bedding beneath the cage. Figure 3 shows the number of high-protein pellets that were actually eaten by hamsters during the various phases of the study. Overall, PFMD hamsters ate significantly more pellets than did NCMD hamsters, F(1, 10) 5 22.2, p , 0.001 (PFMD: 87.3 6 6.11 pellets/day; NCMD: 49.0 6 5.38). Furthermore, a significant maintenance diet 3 ratio requirement interaction, F(5, 50) 5 4.20, p , 0.05, reflects the fact that increases in the FR requirement had dramatically different effects on pellet consumption for hamsters on the two maintenance diets. When housed in their home cages during the initial phase of the study with high-protein pellets freely available, hamsters on the two maintenance diets ate similar numbers of pellets. However, as the fixed ratio requirement increased, NCMD hamsters demonstrated a dramatic decrease in their pellet consumption, and by the end of the experiment, they were consuming only about 10% as many pellets as they had at the start. In contrast, as the fixed ratio requirement increased, PFMD hamsters only slightly decreased the number of high-protein pellets that they consumed, and they ate significantly more high-protein pellets than did NCMD hamsters at FR3, FR4, and FR5.
Over the course of the experiment, PFMD hamsters derived about 12% less energy from the maintenance diet than did NCMD hamsters, F(1, 10) 5 11.3, p , 0.01 (PFMD: 27.2 6 0.73 kcal/day; NCMD: 30.6 6 0.70). However, the increased consumption of high-protein pellets by PFMD hamsters resulted in there being no significant difference in total energy consumption between the two treatment groups, F(1, 10) 5 0.20, NS (PFMD: 33.5 6 1.02 kcal/day; NCMD: 34.1 6 0.92). Of course, over the course of the experiment PFMD hamsters derived a significantly smaller percentage of total calories from protein than did NCMD hamsters, F(1, 10) 5 233.7, p , 0.001 (PFMD: 13.3 6 0.65%; NCMD: 25.8 6 0.49). Furthermore, although the percentage of protein-derived calories decreased as the FR requirement increased, PFMD hamsters still derived an average of 11–12% of total calories from protein even at the highest FR requirement levels, a level of total protein intake that is either adequate for adult hamsters or very nearly so (1,3). Nevertheless, PFMD hamsters gained significantly less weight than NCMD hamsters over the course of the experiment, t(10) 5 2.31, p , 0.05 (PFMD: 2.8 6 4.1 g; NCMD: 13.8 6 2.5). DISCUSSION
Previous research has shown that hamsters respond adaptively when environmental circumstances make it more difficult than usual for them to obtain sufficient amounts of dietary protein. Specifically, time-restricted access to dietary protein causes hamsters to develop a strong and selective preference for a protein-rich diet that is only intermittently available (5,9,11). A similar pattern of behaviour has also been observed in rats (6,10). The present experiment demonstrates that protein-restricted hamsters that must press a lever to obtain dietary protein also make adaptive behavioural adjustments when they are challenged with increases in the FR requirement. Hamsters maintained on a nutritionally complete diet were found to reduce both their rate of lever pressing and their consumption of high-protein pellets when the FR requirement was increased, and at the highest FR requirement levels, they earned and ate very few pellets. In contrast, hamsters maintained on a protein-free diet increased their rate of lever pressing when the FR requirement was increased. Moreover, even at the highest FR requirement levels, they achieved an adequate level of dietary protein intake,
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although their weight gain was substantially less than that of hamsters maintained on the nutritionally complete diet, presumably as a result of their reduced protein intake. It is noteworthy that hamsters maintained on the nutritionally complete and the protein-free diets consumed similar amounts of high-protein pellets when the pellets were either freely available or when the FR requirement was low. Behavioural differences between hamsters eating the two different maintenance diets became evident only when the FR requirement increased. Thus, the use of the operant conditioning paradigm permitted the detection of substantial motivational differences between hamsters maintained on the nutritionally complete and the protein-free diets. Mammals make use of protein to fulfill a variety of vital functions and to maintain the structural integrity of the body. However, mammals continuously lose protein from the body in the course of everyday metabolism, and they do not possess specific cells or tissues that are distinctly specialized for the task of storing either amino acids or protein. Therefore, protein must be obtained in the diet on a regular basis for the maintenance of good health. Consequently, although the ingestive behaviour of hamsters is characterized by a variety of striking peculiarities (7,8,13–16), it is not surprising to find that hamsters nevertheless resemble rats in responding adaptively both to time-restricted access to dietary protein (6,10) and as shown here, to increases in the workload required to obtain dietary protein in an operant situation (2). Although the design of the present experiment does not allow one to conclude that protein-restricted hamsters maintained their lever pressing because the pellets were protein rich, the findings
of other studies strongly suggest that this is so. Thus, in a recent experiment, hamsters housed in standard cages were at different times maintained on protein-free and nutritionally complete maintenance diets, and they were also allowed intermittent access to protein-rich and carbohydrate-rich test diets that contained distinctive marker flavours. When later allowed to select between the flavoured test diets in a brief preference test, hamsters strongly preferred the protein-rich test diet when they were being maintained on the protein-free diet, but not when they were being maintained on the nutritionally complete diet. Furthermore, when allowed to select between two nutritionally identical diets that differed only in their marker flavour, hamsters showed a strong preference for the flavour previously associated with the protein-rich test diet when they were being maintained on the protein-free diet, but not when they were being maintained on the nutritionally complete diet (11). In light of these findings, it appears likely that the protein-restricted hamsters in the present experiment learned to associate the orosensory properties of the high-protein pellets with the consequences of their ingestion, and that they adjusted their lever-pressing behaviour in order to maintain an adequate level of dietary protein intake as the FR requirement was increased. ACKNOWLEDGEMENTS
This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to David DiBattista. The author is grateful to Dayle Belme, Rosa Brew, Debbie Chindemi, and Traci DiBattista for their technical assistance.
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