Mouse nutrition is a complex and multifaceted topic, especially in preclinical research. Diets have an enormous impact on the overall health and welfare of mice, their development, and changes to their microbiomes, and this has a significant effect on research reproducibility and outcomes. What may appear as a simple statement, “mice need to eat a well-balanced diet,” can quickly become confusing with the number of available options. There are multiple commercial vendors, different diet types, formulations, and sterilization techniques available. Here, we would like to give a brief overview of the rodent diet types, sterilization techniques, and potential research variables that surround diets. All of this information can be found with more detail in Chapter 10 of The Jackson Laboratory Handbook on Genetically Standardized Mice.
There are three main diet types and two different ways to formulate these diets.
All of these diet types can be provided with an open formula, meaning that the nutrients do not vary, and the exact proportion of ingredients is published. Natural ingredient diets are often available as a closed formula diet, where the manufacturer lists the ingredients but not the specific formulation. The formulation will change depending on the ingredient market price and availability. Therefore, protein, fat, and fiber will stay as consistent concentrations between batches, but the specific ingredient types will change.
At JAX, we use several standard mouse diets. For most production colonies, we use a closed formula, custom diet upon the NIH-31, 6% fat (w/w) or 10% fat (w/w) diet (LabDiet® formulations 5K52, 6%; 5K20, 11%). The diets for our production mice are available on the strain datasheets under “Technical Support/Dietary Information.”
To ensure reproducibility and reduce variability, institutions have moved toward housing mice in barrier facilities and maintaining colonies in a specific pathogen-free (SPF) status. Many murine pathogens are known to result in immune perturbations that can affect research outcomes (Hsu, et al., 2016; Macy, et al., 2011). It has recently been shown that the pellet manufacturing process itself does not eliminate all rodent pathogens in the feed (Adams et al., 2019; Kurtz et al., 2018). This highlights the importance of sterilizing feed, as well as any items coming into contact with personnel and the mice. There are two main sterilization techniques available for mouse feed:
The autoclave has been in use and evaluated for its effectiveness for decades (Ford, 1977), using steam and time to disinfect. Sterilization effectiveness depends on load size and the ability to penetrate all surfaces. It is essential to have quality control measures in place when maintaining and operating autoclaves. This ensures that the loads have been properly sterilized, and all machine components continue to work appropriately. At JAX, we prefer to utilize autoclaved diets for our mice as we have extensive process validation and routine monitoring of nutrient quality.
Autoclaves have a high upfront cost and require an initial significant time investment to develop and validate procedures for various load types. However, once these have been established, and the machines are maintained appropriately, autoclaves are very effective at sterilizing feed. It is crucial to validate autoclaves for your operational uses. For instance, if you were to autoclave feed and you want to sterilize whole bags at a time, you would need to validate the machine to handle the number of packs you want to disinfect. A validation process for one method is not guaranteed to transfer to all potential uses. For example, if your facility decides to switch to autoclaving whole cage setups with feed in a hopper, the process would need to be re-evaluated. The same would be true if the facility decided to process fewer bags at a time, as compared to the initial validation. Therefore, even basing your facility’s temperature and time on previously published data may not be relevant for your machine. Here at JAX, we strongly encourage each facility to validate their autoclaves for their unique facility needs.
There are some aspects of diet quality that are affected by autoclaving, such as clumping, which makes it more difficult for the mice to access food within a hopper. Clumping is also known to alter nutrient quality. Autoclaves are known to reduce the availability of heat-labile vitamins, including vitamins A, B12, E, thiamine, pantothenic acid, and pyridoxine (Twaddle, et al., 2004). At The Jackson Laboratory, we utilize an extruded diet, which results in less pellet hardness when autoclaved.
Food can be irradiated via Cobalt 60 gamma rays, electronic beam (E-beam), or X-ray delivery. Rodent diets are commonly irradiated with cobalt 60 or E-beam. This has proven to be effective at eliminating pathogens, similar to autoclaving. However, depending on the starting dose of contamination, irradiation does not guarantee full sterility. A recent study demonstrated that the pelleting process for feed does not eliminate viral pathogens and that autoclaving feed is effective at eliminating murine parvovirus and murine norovirus, specifically. However, the irradiation process did not prevent infection of mice with murine parvovirus if the feed had a high viral load (Adams, et al., 2019). Moreover, the manufacturers who irradiate diets do not guarantee sterility of the product regardless of the irradiation method.
Irradiation dose delivered to diets is measured in the unit Gray (Gy), where a typical diet for barrier-housed mice is often exposed to 25kGy, and diets for germ-free rodents are exposed to 50kGy (Caulfield, et al., 2008). The irradiation process, utilizing any method, does not leave residual radioactivity on the product, ensuring it is safe for mice to consume.
Irradiation does generate effects in the diet that may confound research results, such as an increase in oxidized lipid metabolites and glucosinolates, which may affect studies in mammary cancer or diabetes (Prasain, et al., 2017). Here at JAX, we only use irradiation for diets that cannot be sterilized via autoclave, such as high-fat diets or diets with drug additives. However, all irradiated diets are tested via culture before entering any mouse facility.
In conclusion, it is best to evaluate diets and utilize the proper diet for your research needs and understand the potential variables that may present. The sterilization method of choice may depend on your facilities. Autoclaving offers in house validation and analysis, whereas irradiation occurs at an off-site facility and does not guarantee sterility. For more information on mice diets, please read The Jackson Laboratory Handbook on Genetically Standardized Mice.
Adams SC, Myles MH, Tracey LN, Livingston RS, Schultz CL, Reuter JD, Leblanc M. 2019.Effects of Pelleting, Irradiation, and Autoclaving of Rodent Feed on MPV and MNV Infectivity. J Am Assoc Lab Anim Sci. 58: 1-9. PMID: 31391143 DOI: 10.30802/AALAS-JAALAS-18-000142
Caulfield CD, Cassidy JP, Kelly JP. 2008. Effects of Gamma Irradiation and Pasteurization on the Nutritive Composition of Commercially Available Animal Diets. J Am Assoc Lab Anim Sci. 47: 61-66. PMID: 19049256
Ford DJ. 1977. Effect of Autoclaving and Physical Structure of Diets on their Utilization in Mice. Lab Anim. 11(4): 235-239. PMID: 926752 DOI: 10.1258/002367777780936558
Hsu CC, Piotrowski SL, Meeker SM, Smith KD, Maggio-Price L, Treuting PM. 2016. Histologic Lesions Induced by Murine Norovirus Infection in Laboratory Mice. Vet Pathol 53(4): 754-763. PMID: 26792844 DOI: 10.1177/0300985815618439
Kurtz DM, Glascoe R, Caviness G, Locklear J, Whiteside T, Ward T, Adsit F, Lih F, Deterding LJ, Churchwell MI, Doerge DR, Kissling GE. 2018. Acrylamide Production in Autoclaved Rodent Feed. J Am Assoc Lab Anim Sci 57: 703-711. PMID: 30360773 DOI: 10.30802/AALAS-JAALAS-18-000011
Macy JD, Cameron GA, Smith PC, Ferguson TA, Compton SR. 2011. Detection and Control of Mouse Parvovirus. J Am Assoc Lab Anim Sci 50(4): 516-522. PMID: 21838982
Prasain JK, Wilson LS, Arabshahi A, Grubbs C, Barnes S. 2017. Mass Spectrometric Evidence for the Modification of Small Molecules in a Cobalt-60 Irradiated Rodent Diet. J Mass Spectrom 52(8): 507-516. PMID: 28544323 DOI: 10.1002/jms.3950
Twaddle NC, Churchwell MI, McDaniel LP, Doerge DR 2004. Autoclave Sterilization Produces Acrylamide in Rodent Diets: Implications for Toxicity Testing. J Agric Food Chem. 52(13):4344-9. PMID: 15212490 DOI: 10.1021/jf0497657