Research Newsletter | Fall 2023

One of the most common interactions with research animal species is between animal handler and animal at times of cage change, experimental manipulation, and physical examination. It is well established that these types of interactions, while necessary, are also a source of stress for the animals.¹ For example, active transfer of mice into a cage leads to a higher increase in plasma corticosterone levels compared to passive transfer without handling; similarly, rats react differently depending on cage-change techniques.^2,3 Approaches to and training for animal handling can impact animal welfare, data outcomes, and may contribute to unexplained variation in research findings and reproducibility in certain mouse strains and between sexes.⁴ The standard method of grasping the tail to restrain and move mice continues to be routinely used, despite evidence that it induces stress⁵, therefore, it is recommended that less aversive, non-aversive, and low-stress handling methods be incorporated into animal research practices.

Gentle handling of laboratory mice has been demonstrated to positively affect rodent behavior as well as interactions with experimenters.^6-8 Use of inanimate objects to transfer rodents between cages and surfaces (e.g., tunnel handling) or gentle scooping and lifting of mice with palms of hands (a.k.a., cupping) without direct physical restraint offer refinements in handling methods that can reduce anxiety-like behaviors and improve the welfare.⁵ Challenges to implementation of tunnel handling have identified concerns about time and cost investments.⁹ Improved welfare using non-aversive handling was demonstrated through modest increases in pup production and reduction of litter losses, indicating associated reductions in stress as well as substantial benefits to breeding operations with only a minor increase in investment of time to manipulate animals without tail-lifting or use of forceps.¹⁰ Video tutorials on gentle handling techniques are available online through the advocacy organization NC3Rs which aids the global research community in aspects of reduction, refinement, and replacement alternatives: https://www.nc3rs.org.uk/mouse-handling-video-tutorial.

Establishing positive interactions with rats is best accomplished in younger pups or newly acquired young animals by manual ‘tickling’ to habituate animals to human interactions prior to assignment to experimental treatments. During this acclimation period, caretakers may expose rats to a series of events that simulate rough-and-tumble play by young rat pups.¹¹ Tickling has been demonstrated to efficiently and practicably reduce rats’ fearfulness of humans and improve animal welfare through reliably modeling positive affective states.⁸

Specific pathologic effects of routine manipulations and handling are typically undocumented, or scientists may be unaware of lesions associated with common manipulations of laboratory mice that require restraint, such as injections, blood sampling, and tumor monitoring. Assenmacher et al. conducted a postmortem assessment of 1,000 mice used in research with 864 animals being heavily manipulated and 136 being handled only for routine husbandry procedures. For those animals with extensive manipulation, this was defined as necessary and repetitive physical restraint, or due to routine injections and manipulations throughout experiments. Osteoarticular lesions were found in ~7% (61 mice) of heavily manipulated mice, and in a single unmanipulated mouse, demonstrating a highly significant association between heavy handling of mice and the presence of traumatic lesions.¹²

Manual grasping of laboratory mice with restraint devices (e.g., forceps), while historically deemed to be of benefit from a biosecurity perspective, very likely leads to subclinical lesions in mice, up to and including bony fractures, soft tissue injury, and potential for unrecognized and untreated pain and distress, which can confound experimental outcomes. The University of Pennsylvania and University Laboratory Animal Resources (ULAR) removed handling forceps from animal facilities in December 2021 in favor of gentle manual restraint. Training of animal handlers in refined and gentle techniques for experimental mice is critical to improved animal welfare and research outcomes and has been cited as the primary area for welfare improvements based upon a survey of laboratory animal veterinarians.^12,13

Aside from handling methods, it has been shown that male-associated olfactory stimuli induce a stress response in laboratory mice and rats, leading to blunted indicators of pain behavior and increased indicators of anxiety.^14,15 The effect of male personnel on rodents was replicated with olfactory exposure to shirts worn by men, bedding from intact and unfamiliar male mammals, and presentation of human cell secretions (e.g., armpit sweat) from men.¹⁴ Mice demonstrate a preference for the scent of female personnel and an increased stress susceptibility when handled by male personnel.¹⁶

The presence of observers has been shown to inhibit the expression of normal behavior in rabbits, by causing a reduction in activity levels and the duration of exploration behavior.¹⁷ This finding was duplicated in a study of rabbits under observation following surgery, in that the presence of an observer inhibited pain response post-operatively, with rabbits hiding some pain signs related to the affected area.¹⁸ Overall, the ability of research animals to differentiate the sex of human experimenters can have measurable effects on behavioral and/or biological responses. The presence of familiar personnel, with whom animals have interacted positively, has been shown to alleviate anxiety-like behaviors and increase consistency in results from animal tests.¹⁹

In animal research, the personnel that interact with research animals (e.g., animal care staff, veterinary professionals, and research team members) will consistently change throughout the experimental and resting/housing periods until end of study. An improved understanding of these influences of handlers on animal responses will be critical to consider in terms of research findings and general interpretations related to reproducibility in biomedical models of human disease.

*** For further information and refresher courses on animal handling and manipulations, please contact the ULAR Scientist Training staff at ular-tr@pobox.upenn.edu. This resource team can also provide specialized training on aspects of injections and blood collection methods, micro-tattooing, rodent identification, catheterization, anesthesia, aseptic technique, and surgical practices, as well as humane refinements in handling procedures.

1. Balcombe JP, Barnard ND, Sandusky C. Laboratory routines cause animal stress. Contemp Top Lab Anim Sci. 2004;43(6):42-51.

2. Rasmussen S, Miller MM, Filipski SB, Tolwani RJ. Cage change influences serum corticosterone and anxiety-like behaviors in the mouse. J Am Assoc Lab Anim Sci. 2011;50(4):479-483.

3. Meller A, Kasanen I, Ruksenas O, et al. Refining cage change routines: comparison of cardiovascular responses to three different ways of cage change in rats. Lab Anim. 2011;45(3):167-173.

4. Novak J, Jaric I, Rosso M, Rufener R, Touma C, Würbel H. Handling method affects measures of anxiety, but not chronic stress in mice. Sci Rep. 2022;12(1):20938.

5. Henderson LJ, Dani B, Serrano EMN, Smulders TV, Roughan JV. Benefits of tunnel handling persist after repeated restraint, injection and anaesthesia. Sci Rep. 2020;10(1):14562.

6. Gouveia K, Hurst JL. Reducing mouse anxiety during handling: effect of experience with handling tunnels. PLoS One. 2013;8(6):e66401.

7. Nakamura Y, Suzuki K. Tunnel use facilitates handling of ICR mice and decreases experimental variation. J Vet Med Sci. 2018;80(6):886-892.

8. Cloutier S, LaFollette MR, Gaskill BN, Panksepp J, Newberry RC. Tickling, a Technique for Inducing Positive Affect When Handling Rats. J Vis Exp. 2018(135).

9. Henderson LJ, Smulders TV, Roughan JV. Identifying obstacles preventing the uptake of tunnel handling methods for laboratory mice: An international thematic survey. PLoS One. 2020;15(4):e0231454.

10. Hull MA, Reynolds PS, Nunamaker EA. Effects of non-aversive versus tail-lift handling on breeding productivity in a C57BL/6J mouse colony. PLoS One. 2022;17(1):e0263192.

11. LaFollette MR, O’Haire ME, Cloutier S, Blankenberger WB, Gaskill BN. Rat tickling: A systematic review of applications, outcomes, and moderators. PLoS One. 2017;12(4):e0175320.

12. Assenmacher CA, Lanza M, Tarrant JC, Gardiner KL, Blankemeyer E, Radaelli E. Post Mortem Study on the Effects of Routine Handling and Manipulation of Laboratory Mice. Animals (Basel). 2022;12(23).

13. Marx JO, Jacobsen KO, Petervary NA, Casebolt DB. A Survey of Laboratory Animal Veterinarians Regarding Mouse Welfare in Biomedical Research. J Am Assoc Lab Anim Sci. 2021;60(2):139-145.

14. Sorge RE, Martin LJ, Isbester KA, et al. Olfactory exposure to males, including men, causes stress and related analgesia in rodents. Nat Methods. 2014;11(6):629-632.

15. Bateson M. Of (stressed) mice and men. Nat Methods. 2014;11(6):623-624.

16. Georgiou P, Zanos P, Mou TM, et al. Experimenters’ sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor. Nat Neurosci. 2022;25(9):1191-1200.

17. Pinho RH, Leach MC, Minto BW, Rocha FDL, Luna SPL. Postoperative pain behaviours in rabbits following orthopaedic surgery and effect of observer presence. PLoS One. 2020;15(10):e0240605.

18. Pinho RH, Justo AA, Cima DS, et al. Effects of Human Observer Presence on Pain Assessment Using Facial Expressions in Rabbits. J Am Assoc Lab Anim Sci. 2023;62(1):81-86.

19. van Driel KS, Talling JC. Familiarity increases consistency in animal tests. Behav Brain Res. 2005;159(2):243-245.