There are many factors that require consideration when designing any preclinical study, such as rodent model selection, housing conditions, treatment dosing schemes, and diet1. Exact specifications decided upon may, in some cases, be driven by availability or standard practices in the selected vivarium. In the case of lab animal diets, for example, it is typical of a vivarium to stock a particular diet and charge researchers for usage as part of the daily cage per diem. Selecting the standard vivarium diet for your research may seem like a logical and economical choice, however, making this selection arbitrarily could have major implications in the future. Although all lab animal diets are formulated to meet basic nutritional requirements (the major exception being if dietary manipulation is a factor in your study), the types of ingredients used can be quite different2. These seemingly small differences could introduce undesirable and unintentional variability in your model in certain instances. Differences between diet types and examples of how they may influence study outcomes will be briefly reviewed in this Insight.
Lab Animal Diet Types
Grain-Based Diets
There are two main categories of lab animal diets: grain-based diets and purified-ingredient diets. General differences are highlighted in Table 1. Grain-based diets, often referred to in the literature as 'standard diet', 'normal diet', or 'chow diet', are typically stocked by animal facilities. These diets are inexpensive and comprised of plant-based ingredients such as soybean meal and alfalfa meal as well as animal byproducts like fish meal that can contribute more than one nutrient to the diet formulation. The nutrient composition can fluctuate within individual ingredients due to environmental factors and can subsequently induce variability between lots of the same diet1,2. For this reason, it may be prudent in some circumstances such as long term studies to reserve a specific lot of diet in your facility for a particular study. Similarly, if you intend to add a compound to a grain-based diet (for example, a drug), it is good practice to ensure that the compound-added diet and the control diet are made from the same lot. Taconic maintains most of its inventory on the NIH-31M grain-based diet, which contains a slightly elevated fat content (through the addition of soybean oil) to support optimal reproduction.Table 1. Differences Between Grain-Based and Purified Diets
| Grain-Based Diet | Purified Diet | |
| Formula | Closed | Open |
| Ingredients | Unrefined: plant material, animal byproducts | Refined: casein, corn starch, soybean oil, lard |
| Batch-to-batch variability? | Yes | No |
| Fiber Characteristics | 15% or more, soluble and insoluble types | Typical formula contains 5% cellulose only |
| Contains anti-nutrients? | Yes - heavy metals, mycotoxins, phytoestrogens can be present | No |
| Autoclavable? | Yes | No; gamma irradiation can be used as an alternative |
| Possible to add drugs/compounds? | Yes | Yes |
| Possible to manipulate micro/macronutrients | No | Yes |
Due to the nature of ingredients, grain-based diets may contain anti-nutrients (mycotoxins and pesticides) as well as ingredients that have the potential to confound your study (polyphenols and phytoestrogens)3. Moreover, dietary manipulations are restricted to simple compound additions; given that individual ingredients in these diets contribute multiple nutrients, it is not possible to perform complex dietary manipulations such as removal of specific minerals or elevating fat content. While it may be tempting to simply add a new fat source on top of a grain-based diet, this practice dilutes the diet of essential vitamins and minerals, leading to unintentional and potentially confounding nutrient deficiencies. If your study design requires careful control of nutritional elements in the diet, then it is best practice to use a purified diet rather than a grain-based diet.
Purified Diets
Purified diets are compositionally defined and are made only with refined ingredients1. Taconic's Diet Induced Obese (DIO) B6 and Diet Induced NASH B6 models are maintained on well-characterized purified diets. These diets are formulated with refined ingredients, each of which contributes only one main nutrient (e.g., casein as a protein source, lard as a fat source). For these reasons, variability between batches is greatly reduced compared to grain-based diets, and there is less concern with using multiple lots within a study2,4. In general, purified diets are more expensive than grain-based diets and cannot be autoclaved. Irradiation is implemented to reduce bacterial load, but it does not guarantee complete sterility5.One of the major advantages of using a purified diet for research purposes is the ability to precisely control and manipulate nutritional elements. It is possible, for example, to remove vitamin A or manipulate the fatty acid profile of a specific diet without altering any other nutritional aspects. Moreover, control diets can be formulated to ensure that the only difference from the experimental diet is the nutrient of interest. This is particularly useful in preclinical studies involving metabolic disorders, where dietary manipulations (e.g., sucrose/fructose addition, cholesterol addition, fat additions) are commonly used6.
Formulating high-fat or high-sugar diets with purified ingredients offers several advantages. Firstly, as mentioned above, each purified diet is formulated from scratch using refined ingredients, rather than by adding additional fat or sugar 'on top' of an existing grain-based diet. This helps to prevent any micronutrient deficiencies that may arise from diluting a grain-based with additional ingredients. Secondly, purified diets can be designed in such a way that the control diet and the experimental diet are formulated with the same types of ingredients but different concentrations. For example, 45 kcal% fat diet and a matched 10 kcal% fat diet can be formulated with the same fat sources, ensuring that the fatty acid profile of the diets is consistent while also inducing weight gain in the high-fat version. Finally, purified diets in a metabolic disorder series can be formulated so that the total calories in the diets are matched, despite differences in concentrations of specific macronutrients. This is important as rodents on a high-fat diet tend to eat less food on a weight basis compared to rodents on low-fat diets.
Dietary Fiber: A Prominent Differentiator of Rodent Diets
Although purified diets offer many advantages with respect to nutritional modulation and control in preclinical models, they do have several major shortcomings compared to grain-based diets, particularly related to metabolic and gut health. The original purified diet, AIN-76A, was formulated to contain 50% sucrose by weight, which has been known to cause metabolic disturbances when administered long-term; modernized formulas have been developed with less or no sucrose, though AIN-76A is still widely used today7. Long-term usage of a purified diet even with low sugar, high starch, and low fiber has been shown to alter glucose metabolism relative to grain-based diets2. Grain-based diets typically do not have elevated levels of sucrose and therefore do not bear the same concerns with long-term administration.Historically, purified diets have also been formulated to contain 5% insoluble dietary fiber in the form of cellulose. The rationale for this choice was due to the fact that cellulose was found to be ill-tolerated by rodents and therefore could only be added at low levels2. In contrast, grain-based diets can contain upwards of 20% fiber (both soluble and insoluble varieties) from different plant sources4. Recent evidence suggests that the unrefined nature of grain-based diets is advantageous for rodent gut health; it is typical for mice on a grain-based diet to have healthier colon morphology (higher cecum weights, longer colon lengths) compared to mice fed a purified diet. In fact, a recent study demonstrated that these changes to colon morphology can occur in as little as two weeks after switching from a grain-based to purified diet8. Purified diets also do not appear to support microbial diversity to the same extent as grain-based diets, which is likely due to the reduced concentration of dietary fiber and exclusion of soluble fiber. Preliminary evidence suggests that the increasing total fiber concentration and including soluble fiber, such as inulin, to a purified diet may improve colon morphology and microbial diversity, allowing for these parameters to more closely resemble animals fed grain-based diets8. Still, more work is required to develop a purified diet that supports long-term metabolic and gut health in rodents to the same extent as grain-based diets.
While it may seem logical to conclude that all purified diets should be reformulated with increased fiber to improve gut health, this may not always be a good idea. For example, the addition of inulin to a high-fat diet blunts weight and fat gain while cellulose and guar gum do not, which, in the context of a diet-induced obesity study could thoroughly skew result3,9,10. It is therefore essential to always consider the choice of diet before beginning any preclinical rodent study.
Conclusions
Grain-based diets and purified diets are inherently different and each offer unique advantages and disadvantages. Generally speaking, it is good practice to utilize the same type of diet for treatment arms in the same study, and additionally, it may be worthwhile to consider quantities required to complete an entire study and secure diet lots accordingly. Purified diets offer great versatility and control with respect to nutrient manipulation, but their usage may have implications with respect to long-term animal health and the gut microbiome compared to grain-based diets. Nevertheless, diet should always be considered and chosen with care for each preclinical study.
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