In this chapter we discuss the basic components of various mouse diets, ancillary factors influencing the effects of diets, and the composition of the diets used at The Jackson Laboratory.
We have learned that certain associated factors should be considered when designing an experiment because they may affect the outcome. If experimental mice are born and reared on one diet, there should be a period of adjustment when a different food is used. The type of feed formulation may be important also. In open-formula commercial feeds the stated nutritional analysis may be unchanging, but the source and, therefore, the quality of the ingredients may vary depending upon the season, availability, and market prices of these ingredients. Though such a diet may appear constant by chemical analysis, the form in which the nutrients are actually available to the animal may be variable. For example, the protein content may have quite different nutritional value, depending upon the type of protein (biological source), certain plant proteins being of much less value than animal proteins as a source of energy. In this chapter we present formulations for four diets which have proved best in reproductive performance and health in the strains for which they are used. The diets and some representative strains are: Diet No. 1 for strains C57BL/6J, A/J, CBA/J, Diet No. 2 (high protein - low fat) for strains DBA/2J, A/HeJ, C57L/J, DBA/1J; Diet No. 3 (low fat) for strains DBA/2J, AKR/J; Diet No. 4 (modified from Morris's, 1944, diet) for good reproduction in most strains, though it tends to increase the deposition of body fat in reproductively inactive or older mice (Hoag and Dickie, 1960, 1962; Fenton and Cowgill, 1947; Lee et al., 1953).
In general, vitamins are not synthesized by the animal body. The amounts of these essential ingredients in a diet are dependent upon availability for absorption in the mouse, presence or absence of antagonists, and balance or equilibrium with other ingredients, although little quantitative information is available about this presumed "balance" ( Albanese, 1963).
Estimates of vitamin requirements of the mouse vary widely among investigators. Amounts of vitamins in feeds have a great range ( Bell, 1962; Cuthbertson, 1957; Lane-Petter, 1963; Porter and Lane-Petter, 1962; Spector, 1956). The ingredients used in the Jackson Laboratory diets are given in Table 5-1 and the recommended amounts of vitamins and the calculated amounts in these diets are given in Table 5-2.
Recommendations as to the quantities of vitamins to be included in mouse diets are unfortunately based on limited evidence. Not only must the diets be formulated to supply the calculated amounts, but also they should be shipped or stored under conditions which minimize deterioration of vitamins. Sometimes additional amounts of vitamins are provided to counteract deterioration. Unfortunately, it is not known what effect excess vitamin consumption will have on mice. Our diets supply an adequate amount of these vitamins if the feed is used within 12 weeks of manufacture, although we prefer to avoid storage beyond 30 days.
Proteins act as biocatalysts or as structural elements in every metabolic process. The stated requirements for vitamins, minerals, carbohydrates, calories, and fats are, therefore, of little importance unless expressed in terms of general dietary protein levels. High protein levels can compensate for low or borderline levels of other dietary ingredients; for example, with low-calorie diets increased amounts of protein serve as a substitute energy source ( Bosshardt et al., 1950; Frazer, 1961; Albanese, 1963). Conversely, one may use lower protein levels when higher amounts of other components are supplied. Little information is available about specific amino acid requirements of mice, but the few studies reported indicate that they are similar to those of the rat ( Bell, 1962; Morris, 1944; Spector, 1956).
Bell ( 1962) found no differences in growth rates of mice (strains not specified) at levels of 17, 19, and 20 per cent total dietary protein. We compared 17, 19, 21, and 23 per cent protein and found differences in reproductive performance in strains AKR/J, DBA/2J, and C57BL/6J ( Hoag and Dickie, 1962). C57BL/6J mice produced more litters when given a diet with 23 per cent protein, whereas strains DBA/2J and AKR/J produced more litters with 19 per cent protein, provided the fat level remained constant. Most available diets contain 17 to 30 per cent protein. Levels as low as 16 per cent have been satisfactory in a few inbred and noninbred mice. Table 5-3 gives the dietary protein levels and other components of the feed in use (1965) at The Jackson Laboratory.
Unsaturated fatty acids, such as linoleic and linolenic, are dietary requirements of mice ( Decker et al., 1950; Bell, 1962; Morris, 1944). Since most animals fats are saturated the source of the fat component for feeds should be noted. In the rat an interdependence exists even between the unsaturated fats, so that combinations provide a more effective protection against X-irradiation injury than does a large amount of a single fatty acid ( Cheng et al., 1954). These studies suggest that the source of dietary fat in radiation experiments should be considered, since radiation sensitivity in rats can be altered by changing the fatty acid levels.
We have found that certain strains of mice such as C57BL/6J exhibit improved reproductive performance on 11 to 12 per cent dietary fat levels (using corn oil as the source), whereas other strains (DBA/2J, C3H/HeJ, and AKR/J) have an improved reproductive performance with only a 6 per cent fat level. Production of B6D2F1 and C3D2F1 hybrids is increased when the high-fat feed is used, even though one or both parental strains have a better within-strain reproductive performance on the low-fat diet.
In our experiments with different levels of dietary fat, using strains DBA/2J and AKR/J, total reproductive performance (numbers of fertile matings, litters, and mice born and weaned) was significantly improved with a low fat level (6 per cent). There was further improvement when wheat-germ meal was added to the low-fat feed.
During their active reproductive period mice can utilize large amounts of unsaturated fats and will eat more of a diet supplying these ingredients. When reproduction ceases such animals may become obese. It has been suggested that the obesity may cause cessation of reproductive activity, but many males retain their reproductive capability. The obesity may be concurrent with reproductive inactivity rather than its cause.
Quantitative studies indicate that mice need calcium, phosphorus, manganese, iron, zinc, and potassium ( Bell, 1962; Lee et al., 1953). The amounts used in our diets are given in Table 5-3. The mouse undoubtedly requires certain other trace minerals but specific needs have not been reported. Most feeds probably supply levels of such trace elements sufficient to meet these requirements.
The adequacy of a diet is determined by the animal's physiological status, metabolic rate, age, length of time it is to be kept, and its genetic constitution. A diet adequate for maintenance of body weight of an adult confined in a small cage may be completely inadequate for one kept in a larger cage. A diet adequate at one ambient temperature and humidity may be inadequate at another. It has been shown that rats require an increased amount of thiamine and choline when maintained at high temperatures ( Mills, 1943).
A diet's adequacy often varies, depending upon the nature of the experiment. Schneider ( 1956) demonstrated a "salmonella resistance factor" in mouse feeds containing large quantities of wheat; this would be an important consideration when designing experiments on salmonellosis. Other investigations demonstrate the important role of diet in resistance or susceptibility of mammals to radiation, viruses, bacteria, and drugs ( Schneider, 1960; Simon, 1960). Adequacy, therefore, must be defined in terms of experimental designs and the goals to be achieved.
There are many reports of deficiency syndromes produced by experimental diets lacking a specific ingredient, but such conditions are rarely encountered in the average colony. The problems usually encountered are malnutrition or dietary imbalances which are often masked by secondary microbial infections and occasionally by inherent anatomical defects. Dietary imbalances may be encountered only after several generations of feeding. We studied an infertility problem in strain DBA/2J manifested only after 2 years (8 to 15 generations) on Diet No. 4 ( Table 5-1). When the strain was first put on the diet, weaning weight increased and reproductive performance improved. But after 2 years, although the weights at weaning remained the same as when the dietary change was initiated, infertility increased to over 50 per cent of the matings, the number of litters per female was reduced, and the number of spontaneous abortions increased. After investigation of microbiological, endocrinological, pathological, and toxicological aspects of the problem, it was found that lowering the fat level and adding wheat-germ meal corrected infertility so that 85 to 90 per cent of breeding pairs became productive.
Finally, adequacy depends also on the manner in which the diet is presented to the animal, i.e., form, hardness, and palatability of feed pellets.
In early studies using inbred mice we considered a 12- to 14-week period an adequate test of a new diet. The investigations were principally concerned with body weight and reproductive performance. However, experience with strains DBA/2J and AKR/J has shown that we must include not only the reproductive performance of the original experimental and control animals, but also the data through at least three generations. Mice may react to any change in diet with either an increase or decrease in weight or reproductive performance, but this effect may disappear after a few generations. It is also evident that dietary studies are conclusive only for a given strain of mice.
The investigator and the animal breeder must both be concerned with the nutrition of experimental animals. Reproduction and growth are not the only criteria for evaluation of adequacy of diets; dietary factors and their role in health and in disease should be given consideration in any experiment. Nutritional imbalance can be as important as dietary deficiency, so that an excess or deficiency of a particular essential can either in itself produce pathological signs or interfere with the utilization of other essential components.
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