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It is important to note that these observations did not result from experiments that were intentionally designed to evaluate housing conditions and order of testing on specific behavioral measures on testing devices such as the elevated plus-maze

Recommended housing conditions and test procedures can interact to obscure a significant experimental effect.

Behavior research methods, no. 4 (2005): 651-656

Cited by: 2|Views6
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Abstract

Routine animal husbandry variables, such as group housing of mice and the order of testing of cage-mates, are currently viewed to be essentially neutral with respect to the outcome of most, if not all, animal-based experiments, including those that utilize behavioral measurements. During the course of experiments that have utilized the el...More

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Introduction
  • Factors outside of any specific experimental manipulation—housing and order of testing of subjects within a specific cage—are believed to be neutral with respect to behavioral measure outcome.
  • The cumulative effect of not recognizing the possible contribution of such factors as housing and order of testing may be that the behavioral effects of the specific experimental manipulation under study, which in the case was subclinical challenge of animals with a gastrointestinal food-borne pathogen, are obscured
  • This investigation was supported by National Institutes of Health Grant MH50431 to M.L. Correspondence concerning this article should be addressed to M.
  • This report concerns itself with the experiences in this research that can aid other investigators in avoiding experimental noise (e.g., Enserink, 1999; Wahlsten et al, 2003)
Highlights
  • Factors outside of any specific experimental manipulation—housing and order of testing of subjects within a specific cage—are believed to be neutral with respect to behavioral measure outcome
  • Prior work has shown that a subclinical infection in mice with C. jejuni results in the production of anxiety-like behavior, as measured on the elevated plus-maze (EPM), without detectable systemic immune activation that otherwise could account for this behavioral change (Lyte et al, 1998)
  • Whereas a significant order of testing effect was observed in the C. jejuni–challenged animals, thereby replicating the results shown in the first experimental set (Figure 2A), it was not observed in the saline-treated controls, because one cage of group-housed mice contained 2 mice that displayed an order effect opposite to that of the rest of the saline group, resulting in a nonsignificant interaction between order of testing and treatment group as determined by a 2 ϫ 2 ANOVA [F(1,16) ϭ 0.69, p ϭ .4182]
  • The present results reveal how sensitive tests of anxietylike behaviors are to the interaction of pair-housing conditions and the consequent experimental procedures for testing the pair
  • It is important to note that these observations did not result from experiments that were intentionally designed to evaluate housing conditions and order of testing on specific behavioral measures on testing devices such as the EPM
  • The interaction and order of testing accounted for more than 50% of the variance
  • These experimental findings were, observed unintentionally and subsequently evaluated for significance in the context of the overall experimental intent to examine the ability of a subclinical bacterial infection to influence behavior
Methods
  • Subjects Thirty-six 5-week-old CF-1 male mice were purchased from

    Charles River Laboratories (Wilmington, DE).
  • All the animals were maintained in the animal facility for 5 weeks to allow for acclimatization before use in experimental protocols.
  • All treatment procedures were approved by the Minneapolis Medical Research Foundation Institutional Animal Care and Use Committee.
  • Per oral challenge of animals with C.
  • Two days before per oral challenge, a stock culture of C.
  • Jejuni (No 29428, American Type Culture Collection, Bethesda, MD) was plated onto BBL Campylobacter agar plates containing
  • Two days before per oral challenge, a stock culture of C. jejuni (No 29428, American Type Culture Collection, Bethesda, MD) was plated onto BBL Campylobacter agar plates containing
Results
  • The interaction and order of testing together accounted for more than 45% of the total variance.
  • The interaction and order of testing accounted for more than 50% of the variance.
Conclusion
  • The present results reveal how sensitive tests of anxietylike behaviors are to the interaction of pair-housing con-

    ditions and the consequent experimental procedures for testing the pair.
  • It is important to note that these observations did not result from experiments that were intentionally designed to evaluate housing conditions and order of testing on specific behavioral measures on testing devices such as the EPM.
  • Instead, these experimental findings were, observed unintentionally and subsequently evaluated for significance in the context of the overall experimental intent to examine the ability of a subclinical bacterial infection to influence behavior.
  • The authors hope the experiences will serve to alert researchers to the importance of such factors and will help them to have open dialogues with their laboratory animal care staff to ensure the balance between animal welfare considerations and the needs of their science
Funding
  • Indeed, the interaction and order of testing together accounted for more than 45% of the total variance
  • The interaction and order of testing accounted for more than 50% of the variance
  • For Figure 3B, the interaction and order of testing accounted for more than 50% of the variance
Study subjects and analysis
5-week-old: 36
These results indicate that special care should to be taken in implementing housing recommendations and that preliminary tests may be necessary to ensure that housing conditions do not interact with tests of the phenomenon under experimental investigation. Subjects Thirty-six 5-week-old CF-1 male mice were purchased from

Charles River Laboratories (Wilmington, DE)
. Upon receipt, the animals were housed at a density of 2 animals per cage (width, 16 cm; length, 22 cm; height, 13 cm) and were placed under a reversed day:night light cycle (dark, 0400–1600 h; light, 1600–0400 h)

5-week-old: 36
As we will reveal below, the effect was to impair experimental efficiency to the point of obscuring the experimental phenomenon under study. Subjects Thirty-six 5-week-old CF-1 male mice were purchased from. Charles River Laboratories (Wilmington, DE)

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