Diet is perhaps the most important factor in experimental animal nutrition. Where the mechanism of action of a particular nutrient is to be resolved, knowledge of the precise composition of the diet for the animal model to be used is of foremost importance. Laboratory animal diets are placed into three categories. One includes cereal-based, unrefined or nonpurified diets whose primary ingredients are provided from natural sources and which are classed as open or closed formula. The composition of the open formula is known and available to the potential user and must be constructed as specified. On the other hand, the composition of the closed formula diets is known only to the commercial manufacturer; thus these diets cannot be expected to have identical nutrient compositions from batch to batch.

Another category of laboratory animal diet is the purified diet. This diet is made of refined ingredients including isolated proteins, refined sugars and oils, and purified sources of vitamins and minerals. The third category is the chemically defined diet. These diets are made of chemically pure sources of amino acids, mono- or disaccharides, and purified fatty acids or triglycerides. Minerals are provided by reagent-grade chemicals, and the vitamins are of the highest purity.

For most studies in experimental animal nutrition, the purified or chemically defined diets are the most widely used. Although the major purified ingredients may contain small amounts of various micronutrients, resourceful manipulation of the ingredients will permit the formulation of diets adequately supplied with all known essential nutrients except the one in question.

In 1977 and 1980, the American Institute of Nutrition (AIN) published the formula of a purified diet for experimental rodents (AIN 1977 and 1980). For the most part, this diet (called AIN-76A) can be easily reformulated to be used in many different types of nutrition studies. As the diet was used over the years, however, numerous problems were discovered. In 1989, participants in an AIN workshop on laboratory animal diets came to the conclusion that the AIN-76A diet needed to be revised. In 1993, diet formulae that can be substituted for AIN-76A were published and given a general designation of AIN-93 (Reeves et al. 1993a).

The criteria used for the AIN-93 formulations were 1) the diets can be made from purified ingredients, 2) they conform to or exceed the nutrient requirements suggested by the National Research Council (NRC 1978 and 1995), 3) they can be made with readily available ingredients at a reasonable cost, 4) the compositions are consistent and reproducible, and 5) the diets can be used over a wide range of applications.

The new diet comes in two versions: AIN-93G to be used during the early growth phase and during reproduction, and AIN-93M to be used during adult maintenance. Table 1 gives the general formulae of these diets. Table 2 gives the formulae of the mineral mixes for each diet, and Table 3 gives the ingredient list for the vitamin mix to be used for either diet. In the reformulation of AIN-76A to AIN-93 diets, some major changes were made. These changes are highlighted below.

Table 1. Formulation of the AIN-93G diet for rapid growth and the AIN-93M diet for maintenance of rodents when casein is used as the protein source1 [View Table]

Table 2. Minerals mixes that supply the recommended concentrations of minerals for the AIN-93G and AIN-93M diets1 [View Table]

Table 3. AIN-93-VX vitamin mix recommended for use with the AIN-93G and AIN-93M diet formulations1 [View Table]

CARBOHYDRATES

PROTEIN

The major limitation of casein is its shortage of sulfur amino acids, particularly cystine/cysteine. To meet the requirements for sulfur amino acids in the AIN-93 diets, the use of L-cystine is recommended. Most other experimental diets use DL-methionine instead of L-cystine. In those diets, it is assumed that the conversion of methionine to cysteine in vivo will be sufficient to prevent a deficiency of this amino acid. When casein is used at 200 g/kg and L-cystine at 3.0 g/kg diet, sufficient concentrations of each essential amino acid will provide the 1978 NRC requirements for growth (NRC 1978). One hundred forty grams of casein and 1.8 g of L-cystine in the AIN-93M diet will provide more than enough of the sulfur amino acids for maintenance of adult rodents, based on the 1978 NRC report.

The fourth revised edition of the NRC Nutrient Requirements of Laboratory Animals (NRC 1995) recommends an increase in the dietary concentration of many of the essential amino acids, including the sulfur amino acids. The estimated requirement for the sulfur amino acids was increased from 6.0 to 9.8 g/kg diet. At 200 g/kg AIN-93G diet, casein falls short of the recommended amounts of these amino acids. Even with the addition of L-cystine at 3.0 g/kg diet, it remains below the recommended amount for growth. To bring the AIN-93G diet up to the new requirement, an additional 1.6 g sulfur amino acid/kg diet will have to be added to the 3.0 g/kg of L-cystine already present. This can be done by using either L-cystine or L-methionine. However, L-methionine might be the amino acid of choice, because studies have shown that high concentrations of L-cystine in the diet of rats increase the chance of a negative interaction with copper metabolism and of lowering the copper status of animals, especially when copper is limiting (Kato et al. 1994).

Because it is sometimes necessary to change the composition of diets to meet experimental conditions, casein may not be the protein source of choice. The investigator should be aware, however, that the use of other protein sources will require alterations in the basic dietary ingredients. For more information about this point, see the reference by Reeves (1996), which describes how to manipulate the dietary ingredients to achieve a balanced diet when various sources of protein are substituted for casein in the AIN-93 diets.

FAT SOURCES

ANTIOXIDANTS

FIBER

VITAMINS

MINERALS

Other changes in the mineral composition of the AIN-93 diets compared with the AIN-76A diet are noteworthy. The 1978 NRC requirement for manganese was 50 mg/kg diet; however, more recent evidence suggests that the requirement is much lower. The 1995 NRC lowered the manganese requirement to only 10 mg/kg diet, which is in agreement with the AIN-93G and AIN-93M diets. Although molybdenum is considered an essential mineral because of its involvement in the metabolism of sulfur compounds (Wang et al. 1992), the 1978 NRC did not have a requirement level, nor did the AIN-76A diet have molybdenum included in the mineral mix. The 1995 NRC recommends 150 µg molybdenum/kg diet, which is in line with the amount proposed for the AIN-93 diets. Chromium was listed in the 1978 NRC as a required mineral, but subsequent investigations could not corroborate earlier suggestions that chromium was essential for rats (Flatt et al. 1989, Holdsworth and Neville 1990); therefore, the 1995 NRC placed it in the "potentially beneficial nutrient" category until more conclusive evidence is found. Chromium is still included in the AIN-93 mineral mixes, but at a reduced amount compared with that in the AIN-76A diet. The selenium concentration in the diet was increased from 0.1 to 0.15 mg/kg, and the source of selenium was changed from selenite to selenate.

The ultratrace elements, other than selenium, molybdenum and iodine, represent a special class of proposed nutrients. When the formulae of the AIN-93 diets were proposed, it was recommended that a group of ultratrace elements be added to the diets. These included fluoride, boron, vanadium, nickel, lithium and silicon (regarded as a trace element). Although to date there is no convincing and/or repeatable evidence that these elements are essential for any specific metabolic process, studies show that they may interact with other nutrients under various conditions to give the appearance of beneficial effects (Nielsen 1987a, 1987b, 1991 and 1995, Seaborn et al. 1991). These elements are found in relative abundance in the natural ingredient diets, but their concentrations in purified diets are often very low. Therefore, minimal amounts were placed in the AIN-93 diets. It is in the best interest of the investigator to factor in and/or minimize fluctuations in the concentration of any dietary component.

PITFALLS