Jun 23, 2026

Ice Test Protocol for Rabbits

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lym31wlx9/v1
  • Brendan Moline1,2,
  • Landon Genry1,3,
  • Cassandra Kramer1,3,
  • Elvia Mena Avila1,2,
  • Emily Reedich1,2,
  • Megan Detloff4,
  • Katharina Quinlan1,2
  • 1George and Anne Ryan Institute for Neuroscience;
  • 2College of Pharmacy, University of Rhode Island;
  • 3Interdisciplinary Neuroscience Program, University of Rhode Island;
  • 4Drexel University
Katharina Quinlan
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lym31wlx9/v1
Protocol Citation: Brendan Moline, Landon Genry, Cassandra Kramer, Elvia Mena Avila, Emily Reedich, Megan Detloff, Katharina Quinlan 2026. Ice Test Protocol for Rabbits. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lym31wlx9/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 19, 2026
Last Modified: June 23, 2026
Protocol  Integer ID: 319423
Keywords: ice test protocol for rabbit, ice test protocol, responsiveness to an aversive cold stimulus, aversive cold stimulus, behavioral test, rabbit, female rabbit, von frey testing, responsiveness
Funders Acknowledgements:
Katharina Quinlan, Megan Detloff
Grant ID: R01 NS135580
Abstract
We use this behavioral test to measure responsiveness to an aversive cold stimulus. Neonatal, juvenile, and adult male and female rabbits can be tested using these methods using equipment very similar to that needed for von Frey testing (enclosures that are open on the bottom, with rabbits resting on a plastic / metal wire grid).
Materials
1. Acrylic restraint box.
2. Raised metal grid.
3. Non-slip mesh.
4. Stopwatch.
5. Ice pokers.
Made with deionized water, cotton swabs, and 500 µL microcentrifuge tubes.
6. Heated nest box.
Safety warnings
Make sure the kit is warm during testing. If the kit feels cold to the touch during testing, pause testing and place kit in a warm nest box until it is warm and ready to continue testing.
Ethics statement
This protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Preparation
Prepare ice pokers at least one day prior to testing by placing the cotton tip of a cotton swab into a 500 µL microcentrifuge tube filled with deionized water. Freeze at -20°C.
Prepare the testing apparatus by placing a soft non-slip grid on a raised metal grid. Place an acrylic restraint box on top of the non-slip grid.
Testing
At the start of testing, place the rabbit kit inside the restraint box. Let it acclimatize and settle (i.e. no constant movement, digging, rearing, etc.) with all four paws flat on the grid.
Be sure to monitor the temperature of the kit over the course of testing. If the kit feels cold to touch, pause the test and return to a heated nest box to raise its body temperature. Resume testing when the kit is warm.
Make sure the kit has not fallen asleep by lightly rubbing/prodding it with your finger prior to data collection.
Retrieve an ice poker from the freezer and remove it from the microcentrifuge tube.
With the poker, approach target paw from beneath at a 90° angle. Aim for the palmar/plantar region just proximal to the metacarpals/metatarsals.
Do not poke between the metacarpals/metatarsals, or near the base of the paw.
Contact the target region of the paw with the tip of the poker and start the stopwatch.
Continue contacting the paw until either the kit has produced a response or after 20 seconds have elapsed without eliciting a response. Write down the time to response and the type of response, or 21 seconds if there was no response.
Responses include:
Withdrawing the paw.
Shaking the paw.
Thumping the paw.
Licking the paw.
General sudden movement (kicking, jumping, falling over, etc.)
Repeat steps 4-7 with the remaining paws. Only one measurement is needed for each paw.
Replace ice pokers as needed if the ice has melted significantly.
Protocol references
Brenner, D. S., Vogt, S. K., & Gereau, R. W., 4th (2014). A technique to measure cold adaptation in freely behaving mice. Journal of neuroscience methods, 236, 86–91. https://doi.org/10.1016/j.jneumeth.2014.08.009

Brenner, D. S., Golden, J. P., Vogt, S. K., & Gereau, R. W., 4th (2015). A simple and inexpensive method for determining cold sensitivity and adaptation in mice. Journal of visualized experiments : JoVE, (97), 52640. https://doi.org/10.3791/52640