feeding of diets of lower nutrient density without decreasing nutrient intakes. High temperature, disturbing stimuli, social conflict, or other environmental factors that reduce food intake may necessitate diets higher in nutrient concentrations to maintain adequate nutrient intakes.

Housing types can also affect the amounts of nutrients needed in diets. For example, laboratory rodents maintained in either galvanized cages or cages with solid bottoms may have a lower dietary requirement for zinc because of the availability of zinc from the feces and cage materials. Solubilized minerals in drinking water (such as copper from copper water lines) may affect the amounts of these minerals that must be supplied by the diet. If laboratory animals ingest bedding or other "nonfood" materials, these may provide an unintended source of some nutrients or toxins. In studies of the requirements of laboratory animals for constituents that might be needed at extremely low concentrations, even the air supply may be a significant source of contamination.

MICROBIOLOGICAL STATUS

Under normal rearing conditions, laboratory animals harbor populations of microorganisms in the digestive tract. These microorganisms generate various organic constituents as products or by-products of metabolism, including various water-soluble vitamins and amino acids. The extent to which these nutrients contribute to the nutrition of the host may be substantial but varies according to species, diet composition, and rearing conditions. In the rat and mouse, most of the microbial activity is in the colon, and many of the microbially produced nutrients are not available to the host unless feces are consumed, as is common for rats and other rodents (Stevens, 1988). Prevention of coprophagy may require an increase in the nutrient concentrations that must be supplied by the diet. The loss of some or all microbial symbionts in animals free of specific pathogens and germ-free animals, respectively, may also alter microbial nutrient synthesis and, thereby, influence dietary requirements. Adjustments in nutrient concentrations, the kinds of ingredients, and methods of preparation must be considered when formulating diets for laboratory animals reared in germ-free environments or environments free of specific pathogens (Wostmann, 1975).

RESEARCH CONDITIONS

Experimental procedures may produce stress or otherwise alter food intake. For example, surgical procedures or test substances in diets may lead to anorexia, necessitating the provision of more palatable diets or diets with elevated nutrient concentrations. Experimental protocols that require restriction of the amount of food offered alter the intakes of all nutrients unless dietary concentrations are altered to account for changes in food consumption.

NUTRIENT INTERACTIONS

Alterations in dietary energy density usually cause a change in feed intake. If high-energy diets are used, it may be necessary to increase nutrient concentrations in the diet to compensate for decreased food consumption. Other interactions occur between nutrients, such as competition for absorption sites among certain minerals that share common active transport systems. Thus in formulating diets containing unusual nutrient concentrations, the potential effects on other nutrients must be considered and adjustments made in nutrient concentrations, if appropriate.

FORMULATION OF DIET TYPES

Diet formulation is the process of selecting the kinds and amounts of ingredients (including vitamin and mineral supplements) to be used in the production of a diet containing planned concentrations of nutrients. Choice of ingredients will be influenced by the species to be fed and the experimental or production objectives. Target nutrient concentrations must take into account estimated nutrient requirements, possible nutrient losses during manufacturing and storage (National Research Council, 1973; Harris and Karmas, 1975), bioavailability of nutrients in the ingredients, and potential nutrient interactions.

Various types of diets are available for use with laboratory animals. Selection of the most appropriate type will depend on the amount of control required over nutrient composition, the need to add test substances, potential effects of feed microbes, diet acceptance by the animals, and cost. Wastage is also a problem with some types of diets, which may be a disadvantage if quantitative intake is to be measured.

The ideal diet for a particular animal colony will depend on production or experimental objectives. The diet must be sufficiently palatable to ensure adequate food consumption and must be nutritionally balanced so that the nutrients essential for the objectives are provided. It should also be free of substances or microorganisms that may be toxic or cause infection. Diets used in research also must be readily reproducible to ensure that the results can be verified by additional studies.

It is common to classify diets for laboratory animals according to the degree of refinement of the ingredients.

NATURAL-INGREDIENT DIETS

Diets formulated with agricultural products and by-products such as whole grains (e.g., ground corn, ground wheat), mill by-products (e.g., wheat bran, wheat middlings, corn gluten meal), high protein meals (e.g., soybean meal, fishmeal), mined or processed mineral sources (e.g., ground limestone, bonemeal), and other livestock feed in-



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