The use of laboratory rodents in basic biological and behavioral research has a long and distinguished history. Rodents are particularly advantageous for research because environmental variables, such as diet, are controlled easily. However, researchers often overlook possible interactions between the rodents' diet and the specific outcome variable. Inappropriate nutrient composition of diets fed to rodents can cause significant variation in otherwise well-controlled investigations. In an effort to assist in the formulation of diets for rodents, the National Research Council (NRC) and the American Institute of Nutrition (AIN) have published several reports listing nutrient recommendations for rats and mice (AIN 1977, NRC 1995, Reeves et al. 1993). The scope of these recommendations is incomplete, however, inasmuch as the nutrient requirements of rats and mice are based largely on data derived from investigations including only immature rodents given access to the experimental diet for short periods. That is, nutritional requirements for mature rodents used in long-term investigations are virtually unknown.

Several areas of research such as oncology, toxicology and gerontology have a critical need for rodent diets providing adequate nutrition over the long term. Nutrient requirements of mice and rats at different stages of development and maturity should be determined by evaluation throughout the life span. The formulation of diets based on testing in weanling rodents may not be appropriate for long-term investigations. Moreover, assumptions and conclusions that apply to young rodents may not have relevance in older animals. Discussion in this review will focus on distinguishing factors related to development of nutrient requirements in young growing rodents from those pertinent to other periods of the life span.

RATE OF GROWTH AND NUTRITIONAL REQUIREMENTS FOR LABORATORY ANIMALS

As useful as growth analysis is to establishing requirements for weanling laboratory rodents, this index is limited because it does not address the nutritional requirements of full-grown adult rodents. The potential problems associated with extrapolating recommendations designed for young growing animals to long-term feeding studies are well illustrated by recent changes in the protein concentration of diets endorsed by AIN. The protein recommendation for the AIN-76A diet (AIN 1977) was established from data describing maximal growth occurring in weanling rats at protein concentrations ranging from 16.7 to 20% (Goettsch 1948). Recent reports suggest that dietary protein concentrations at or near the AIN-76A diet recommendation for extended periods enhance the severity of nephropathy in older rats. Decreasing the protein content from 20% to 13-15% reduces significantly the incidence of kidney lesions (Rao et al. 1993, Yu et al. 1985). In response to these investigations, the ad hoc writing committee on the reformulation of the AIN-76A rodent diet recommended that rats be maintained after rapid growth (although no specific age was given) on diets with a casein concentration of 13% (Reeves et al. 1993). It is somewhat surprising that the recommendation for protein did not include a suggestion for soy rather than casein protein for long-term feeding. The substitution of soy protein for casein seems to significantly reduce kidney lesions in rats fed these diets (Iwasaki et al. 1988a).

The effects that other nutrients may have on the health of rodents fed diets over the long term are less clear than that described for protein. Some investigations suggest that the dietary requirements for carbohydrate and lipid may differ in young and mature rodents (Iwasaki et al. 1988b, Murtagh-Mark et al. 1995). On the other hand, the calcium content of the diet does not seem to affect the incidence of kidney lesions or the life span of F344 rats (Iwasaki et al. 1988b). Further investigation is necessary to elucidate more fully the nutrient requirements in long-term studies.

NUTRITION RESEARCH IN WEANLING RATS AND MICE

The survey described here demonstrating the disproportionate use of weanling vs. mature rodents in nutrition-related research is not intended to suggest that these experimental designs are flawed. Use of the weanling rat as a model to describe the interactions between nutrition and normal biology clearly has a place in animal research. Rather, these examples illustrate a problem that can occur in only lines of investigation, i.e., reliance on any one particular model system to characterize phenomena severely limits the general application of the results.

The use of the weanling rodent as a general model in nutrition investigation is especially problematic when attempts are made to extrapolate the findings to older animals. This point is well illustrated by results from investigations evaluating the interaction among diet, glucose intolerance, insulin resistance and aging. In the late 1970s and early 1980s, several investigations reported that glucose intolerance increased as a function of age (Reaven et al. 1983, Reiser and Hallfrisch 1977, Wright et al. 1983). These findings were consistent with the human data describing increased incidence of noninsulin-dependent diabetes mellitus with age (Kreisberg 1987). Reaven and colleagues described insulin resistance being greater and insulin secretion being less in 12-mo-old Sprague-Dawley rats compared with results observed in 1.5- to 2-mo-old animals (Reaven et al. 1983). Subsequent investigations raised questions as to the relevance of these data to studies of age-related changes in insulin resistance. That is, a 12 mo-old animal represents less than half the median life span for the Sprague-Dawley rat, whereas a 2-mo-old animal is still within the exponential growth phase for this strain. Goodman et al. (1983) observed that results in young rodents may more closely reflect changes that occur because of rapid growth rather than aging per se. These investigators found that glucose removal rate, as measured by euglycemic clamp, is significantly greater in 2- vs. 4-mo-old rats. However, glucose removal does not differ significantly from 4 to 24 mo of age. Several other investigations have also found that insulin secretion (Ruhe et al. 1992, Starnes et al. 1991), glucose tolerance (McDonald 1990) and insulin receptor function (Eiffert et al. 1991) do not differ significantly in rats between 6 to 24-26 mo of age.

The length of time that rodents are fed the experimental diet may also significantly affect the results. For example, Reiser, Hallfrisch and colleagues (Hallfrisch et al. 1979, Reiser and Hallfrisch 1977) reported that weanling Wistar rats fed 54% sucrose (g/kg) from 3 to 11 wk have impaired glucose tolerance and reduced insulin sensitivity of epididymal fat compared with animals fed starch. It is unclear, however, if the findings reported by these investigators reflect a general nutritional effect or an effect of short-term diets. Our laboratory has conducted a series of experiments to evaluate the long-term consequence of feeding 66.0% sucrose (g/kg) on several biological markers. In cross-sectional studies using rats of ages 6, 12 and 26 mo and fed the experimental diet for 4-6 mo, we found no differences in basal plasma glucose and insulin concentration (Hara et al. 1992), in vitro insulin secretion (Hara et al. 1992) or glucose removal rate of perfused hindlimbs (Eiffert et al. 1993) compared with starch-fed rats. Moreover, in recently completed longitudinal studies in which rats were fed 66.0% sucrose (g/kg) throughout their life span, we did not observe differences in several indices of glucose homeostasis compared with starch-fed animals (Lingelbach et al. 1996, Ruhe et al. 1996).

The examples used here imply that both the animals' age and the length of time they are fed the experimental diet can significantly affect results. Generalization made from observations in weanling rodents may not be appropriate for all ages. Diets should be tested in long-term studies using older animals. Nutritional sciencesand I suspect many other fieldswould benefit greatly by including a wide range of age groups and dietary treatments in their model systems.

AD LIBITUM FEEDING AND LONG-TERM CONSEQUENCES

Using longevity to imply that energy intake restriction in rats and mice is the most beneficial dietary practice is valid but simplistic. Longevity and the delay of age-related disease are only two indices of optimal nutrition and should not be used as the only criteria by which dietary recommendations are established. Consideration must be given to the possibility that energy conservation associated with the slower growth rate seen during early development of rats and mice may detrimentally alter specific physiological systems. This possibility is particularly significant in regard to reproductive fitness. Short-term food restriction as well as suboptimal nutrient density of the rodent diets delays significantly the onset of puberty in male and female rats (see review, Depaolo 1994). However, Merry and Holehan (1979) found that, in the long term, energy restriction, not food restriction, did not significantly affect reproductive fitness. Rather, rodents adapt to the energy restriction by delaying puberty but lengthening the reproductive period. The onset of puberty in energy-restricted rats is 40-60 d later, but osestrous cycle irregularities occur significantly later in life that those observed in ad libitum-fed animals.

The results from investigations evaluating the effect of energy restriction on reproductive fitness suggest that normal development may be delayed but not necessarily altered. The delay in development of energy-restricted animals may represent what some have termed the down-regulation of growth (Barker 1996). That is, the rodent may "adapt" to the lower energy intake by requiring less nutrient flux to maintain normal physiological function. McCarter and colleagues (McCarter and McGee 1989, McCarter and Palmer 1992) tested this general hypothesis, in part, by evaluating 24-h energy expenditure in ad libitum-fed and energy-restricted male F344 rats for 24 mo. Energy restriction initiated in weanling rodents suppressed 24-h energy expenditure, expressed per metabolic mass, at 6, 8, 10, and 12 wk of age. However, by 24 wk of age, basal metabolic rate, expressed independent of body mass or as a function of lean body mass, did not differ between ad libitum-fed and calorie-restricted rats. The lack of differences in energy expenditure between the two groups persisted until the end of the experiment when the rats were 24 mo of age.

Further support for the "down-regulation of growth" hypothesis comes from recent findings on glucose utilization in energy-restricted rats. The observation that serum glucose and insulin concentrations are significantly reduced in energy-restricted vs. ad libitum-fed rats led Masoro et al. (1992) to suggest that the enhanced longevity associated with energy restriction reflects alterations in fuel utilization. Although plasma insulin and glucose concentrations were significantly lower in energy-restricted compared with ad libitum-fed rats, there was no difference in the mean 24-h respiratory quotient or total energy expenditure per unit of metabolic mass. That is, there were no alterations in fuel utilization pattern, and the energy-restricted rats simply used less glucose than did the ad libitum-fed rats. Others have shown the energy restriction in older rhesus monkeys results in increased insulin action (Bodkin et al. 1995). Together, the investigations of Masoro et al. (1992) and Bodkin et al. (1995) strongly suggest that smaller, energy-restricted mammals function better without altering the basic physiological response.

The evidence is now unequivocal that energy restriction in rodents increases life span and reduces the incidence of many age-related diseases as compared with ad libitum feeding. These effects are also seen when the energy restriction is more modest than the widely used regimen of 60% of ad libitum intake. Reports have shown that even moderate energy restriction can extend life, delay biochemical and physiological loss, and decrease the incidence of cancers (Weindruch et al. 1986). Indeed, at least one investigation has shown that a reduction in energy intake of only 5% increases activity of aged rodents (Holloszy et al. 1985). Although additional investigations are required to more fully elucidate the long-term effect of energy restriction, it seems reasonable to suggest that the time has come for a re-evaluation of ad libitum feeding as the standard dietary paradigm used in rodent investigations.

CONCLUSION