The Importance of Incorporating Both Sexes and Embracing Hormonal Diversity When Conducting Rodent Behavioral Assays

Abstract

Reproductive hormones such as estrogens, androgens, progesterone, and others are responsible for the regulation of countless body functions in addition to their well-known control over reproductive function and behavior. The list of processes governed by internal hormonal secretions is extensive and, as such, hormonal fluctuations influence how animals perform on many established behavioral measures. Additionally, their pervasive impact can alter the way that non-hormonal drug treatments act across individuals. Male reproductive hormones are relatively stable for most of the young adult life span, and, because of this, males tend to demonstrate less variability in behavioral and physiological assessments. This stability is tempting to focus upon when investigating the behavioral effects of a new therapeutic; however, it has become increasingly clear that the effects of pharmaceutical and other manipulations can depend on the background hormone milieu of the individual. Understanding interactions with hormones is vital for the optimization of any potential or existing therapeutic and, therefore, the inclusion of measures outlined in this chapter should be considered when designing rodent behavior studies for this purpose. This chapter discusses the reproductive system and hormonal profiles of male and female rodents, how these hormonal profiles impact commonly used behavioral evaluations, and techniques for monitoring and manipulating these hormone levels, to produce optimal behavioral assessments in rodents.

Appendix: Vaginal Smear Protocol

Supplies

• Sterile saline

• Small beaker

• Small cotton-tipped applicators

• Microscope (10× magnification with backlight)

• Sheets to record observations

• Plastic apron

Procedure

• Set up supplies:

  • Fill a beaker with ¼ in. of sterile saline and soak cotton-tipped applicators in sterile saline to soften and moisten the cotton.

  • Set up a microscope with a blank slide nearby.

• To vaginally smear a rodent:

  • Pick up a rodent by the base of the tail with the rear end of the animal facing you.

  • Gently grasp the tail with your thumb and forefinger, and lift it away from the rear end of the animal, so that you have a clear view of the anogenital area.

  • Insert a moistened cotton-tipped applicator into the vaginal canal no more than 1 cm, and very gently swab the vaginal walls to collect cells.

  • DO NOT insert the cotton-tipped applicator more than 1 cm into the vaginal opening.

  • Place the animal back into its home cage.

  • Carefully roll the cotton swab onto the blank slide, trying to get as much liquid (and cells) onto the slide as possible.

  • Focus the microscope on the slide and inventory the types of cells present in the smear.

    • Goldman et al. [10] give a description and pictures of the various stages of the rat estrous cycle.

    • Mennenga and Bimonte-Nelson [21] give a description and pictures of vehicle-treated versus estrogen-treated Ovx rats.

  • Record the relative percentage of each type of cell present in the smear and which stage of the estrous cycle the animal is likely to be in.

  • Repeat for at least 4 days for ovary-intact animals (to confirm a cycle) or Ovx animals with or without hormone treatment to ascertain presence of stimulation.

  • Staining and coverslipping procedures are also performed in some cases to more clearly note cell types and preserve smears over time.

Additional Helpful References

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