Feed and Diets

What you should know about reducing experimental variation through proper selection of diet for laboratory rats and mice.

Researchers from all disciplines want to reduce variability in their studies, but they may not recognize that diet is a source of variability^1,2 This article describes some of the factors that personnel involved in diet selection for an institution or for a specific study should be aware of, and how the potential to confound experimental results can be avoided or minimized.

Types of Diets
Diets fed to laboratory animals can be generally classified as one of two types — purified or natural ingredient. Purified diets are made of refined ingredients such as casein, amino acids, sucrose, fructose, corn starch, various fats or oils, cellulose, vitamins, and minerals. Such mixtures are well suited to minimize nutrient variation, certain environmental contaminants,³ and the presence of various non-nutritive biologically active compounds naturally occurring in plants.^4,5 Purified diets are important research tools because they can be manipulated to contain very high or low levels of specific macro nutrients (i.e. 60% of kcal from fat or 6% protein) and micro nutrients (i.e. 2% calcium or vitamin A deficient).Many studies do not require such precise control and an appropriately selected natural ingredient diet is suitable.

The largest volume of laboratory animal diet produced is comprised of agricultural commodities like corn, wheat, plant by-products, soybean meal, oats, alfalfa meal, and animal derived ingredients such as fish meal and meat and bone meal. Unfortunately there is no consistent terminology used across the industry, and these diets might be called “standard” or “grain-based” or “chow.” In this article these types of diets are most correctly referred to as natural ingredient diets, which denotes the relative lack of processing the ingredients have been subjected to.

Natural ingredient diets support reproduction, growth, and maintenance of laboratory animals. There are a variety to choose from, designed for various life stages or a more specific application. A major source of information about a natural ingredient diet is the technical data sheet that a manufacturer makes available, usually via their website or printed materials. While these data sheets are an essential resource, it is also important to have reasonable expectations about the accuracy and precision of the nutrient information. The nutrient levels are estimates, derived from a variety of sources including published commodity compendium data, wet chemistry testing of raw materials, and finished product testing (the latter particularly for protein, fat, and crude fiber). Nutrient losses due to heat treatment and mechanical processes during manufacturing, or post-production effects of irradiation or autoclaving are not routinely taken into consideration in these estimates.

As a general rule, the degree of variation, calculated as the coefficient of variation (CV) for the nutrient classes will be as follows: protein –CVless than 5%; fat –CVup to 10%;minerals –CVgenerally less than 10%, although some such as iron can be more variable; vitamins – CV ranges from 5%to 20% or more. This wide range is due to both variable manufacturing loss and, in some cases, fairly sizeable analytical variation. If the interpretation of an experiment depends on accurate knowledge of a nutrient level, it should be measured; this is advisable whether using a natural ingredient or purified diet.

Looking at the list of ingredients is also instructive. While the actual recipe is rarely disclosed, the major commodity ingredients present in a diet will be listed in descending order of inclusion. When comparing diets on paper, reviewing the ingredient listing and the nutrient information results in a more meaningful evaluation than either alone.

Diet Formulation Philosophy
Diets for laboratory animals can be classified as open or closed. Open formulas are in the public domain, meaning that any manufacturer could produce it and researchers can report a full formula. Examples of these include natural ingredient diets created by government agencies like NIH and NTP. Also, the majority of purified diets are considered open, as most manufacturers of these diets fully disclose these formulations to the end-user. Closed formulas are proprietary, meaning that while the ingredients are disclosed, the exact recipe is known only to the manufacturer; the end-user relies on the technical datasheet for details about the diet. Open formulas are by definition fixed; closed formulas can be either fixed or variable.

Fixed formula diets use the same ingredients, in the same proportion, each time the diet is produced. In contrast, both ingredients and ingredient inclusion rates may be adjusted in variable formula diets. The justification for the use of variable formulas is that adjustments are necessary because raw material macronutrient variability can be of a magnitude to cause significant variation in the finished product. However, the effects of geographical and seasonal variation on ingredient nutrient levels are typically overstated. Let’s take the example of protein since it is on this basis that formula adjustments are likely taking place. In a recent report on quality and variation in soybean meal sources,⁶ the maximum difference in the average protein content of soybean meal from six highly varied countries was 2.8%. To put in the context of finished feed, this would mean an average difference in protein in the finished product of about 0.14% to 0.7%, considering that soybean meal is probably going to be present in many laboratory diets at about 5 to 25%. This slight difference would furthermore be balanced by the protein variation in other ingredients.