• Old mice: In well-maintained mouse colonies, median longevity of most mouse genotypes is 25-30 months. Median longevity of modern humans in medically-advanced countries is ~80 years. These numbers can be thought of as starting points for how to think about the “agedness” of your mice.
• Note that the older the animals you use, the more likely they are to have occult pathologies that may affect your phenotypic outcomes, particularly behavioral outcomes
• Young mice: Mice are physiologically capable of reproduction at 5-6 weeks of age but they are not yet fully adult, any more than 12-year-old humans capable of reproduction are fully adult. Both mice and humans at these ages are still physically and behaviorally immature. Fully mature adult mice in most genotypes will be 3-6 months old.
• Similar considerations, and similar ages, apply to laboratory rats
• For C57BL/6J and UM-HET3 mice, when choosing ages to start experiments or to analyze tissues from “young” and “old” mice, many investigators will use mice approximately 6 months old or 22 months old, respectively.
• The current recommendations are to analyze multiple time points.

Since 2016 the NIH has mandated that researchers consider sex as a biological variable in animal studies. In aging research sex effects have proven to be nearly ubiquitous. However, inclusion of both sexes is not the same as taking sex as a biological variable seriously. Taking sex seriously means including enough animals of each sex to be able to detect a sex difference if there is one.

In recent years, the range of mouse genotypes available for research has expanded dramatically. There continue to be a host of inbred strains available, C57BL/6 is by far the most commonly used. F1 hybrids share the advantage (and disadvantage) that all individuals are genetically identical, but they are more physiologically robust. There are now available a variety of controlled outbred stocks, such as the UM-Het3 and Diversity Outbred stocks. These have the advantage that each individual mouse is genetically unique but the population is genetically controlled. Wild-derived and other genetically uncontrolled outbred stocks are also available. All of these have their strengths and weaknesses. They also differ in a wide range of traits relevant to aging. The important thing is that you should be able to justify the genotype you choose to work with. A good source of information about research mouse genotypes is the Jackson Laboratory Phenome Database (https://phenome.jax.org/).

• Since the 1960’s when many experiments were spoiled by mouse colony disease outbreaks, most commercial and research institutions have relied on Specific Pathogen–Free (SPF) status. SPF colonies are not free of all disease, and are not free of all infectious agents, but they are free of a well-defined group of known rodent pathogens that routinely plague the old-fashioned “conventional” mouse colony. The use of SPF mice has greatly improved the reproducibility of studies among laboratories.
• However, there are also drawbacks associated with the use of SPF animals. Recently, it has become apparent that in protecting our animals from these infectious agents, which we do by excluding as many microbes as possible from their environment, we have also deprived our animals of a normal microbiological experience which can have far-reaching effects. Most obviously, as the microbiome is centrally involved in developing a normal immune system, adult mice from SPF colonies have poorly developed immune systems, more akin to neonatal humans than adult humans.
• The important thing is to carefully consider the microbial status of your mice in light of your planned experiments

• The original gerotherapeutic was dietary restriction. Eating less improves the health of laboratory rodents in most ways we can measure and also increases longevity. Therefore, any treatment whether drug or genetic modification resulting in animals eating less will also make them live longer. It is important not to conflate the dietary effect from the drug or genetic effect. Monitoring food intake is critical.
• Monitoring body weight is not equivalent to monitoring food intake.
• Beware of mixing and matching “chow” diets. Chow diets from different manufacturers differ substantially in their macronutrients.

All of the above are guidelines rather than rules. Researchers must follow the dictates of their particular experimental paradigms, however there is never a downside to keeping these guidelines in mind.

AFAR would like to thank Steven N. Austad, Holly Brown-Borg, Catherine Kaczorowski, and Dudley Lamming for developing these guidelines.

Additional resources:

• Best Practices for Using Animals in Aging Research: Webinar recording from the Nathan Shock Centers of Excellence: https://nathanshockcenters.org/june2021webinar-1.
• Richard A. Miller, Nancy L. Nadon, Principles of Animal Use for Gerontological Research, The Journals of Gerontology: Series A, Volume 55, Issue 3, 1 March 2000, Pages B117–B123, https://doi.org/10.1093/gerona...