TITLE: Mouse Water Restriction
AUTHORS: Avalon Amaya, Jackie Swapp

[DESCRIPTION]
This protocol describes a semi-customized workflow to perform water restriction with mice designated for future experiments. 

Water restriction is a common method used to increase engagement and motivation of mice performing behavioral experiments that use water as a reward.

Water restriction timeline summary: Mice should have three daily weights collected, which are averaged to determine their baseline weight. The target weight will be a percentage (desired) of their baseline weight. The first day of water restriction begins on the last day of the mouses weight collection and is given 1.0mL of water. The mouse should be weighed and provided at least 1.0mL of water daily while under water restriction. All calculations should be individually determined for each mouse. 

Mice under water restriction should be closely monitored for health concerns due to the effects of dehydration. All mice should receive 1.0mL of water daily. Follow IACUC and veterinary requirements for water restricting mice. 

[GUIDELINES]
Only perform this procedure in accordance with IACUC and veterinary requirements.

Animal Behavior:
Utilize a 12-hour light cycle.
Mice should have access to ad-lib food and water until placed on diet restriction. 
Single house mice on diet or water restriction.
Ensure cleanliness of experimental environment.

[STEPS]
SECTION: Maintaining a Mouse on Water Restriction
15. Open Waterlog and access mouse record by repeating   
SECTION: Taking a Mouse off Water Restriction
23. Repeat section "Maintaining a Mouse on Water Restriction". Specifically   .

 

SECTION: Taking a Mouse off Water Restriction
24. Remove mouse from water restriction in Waterlog.
SECTION: Taking a Mouse off Water Restriction
24.1. Click the edit/pencil icon button to remove mouse from water restriction. Confirm this change within the popup window.

 

 

SECTION: Taking a Mouse off Water Restriction
25. Confirm that mouse is to be removed from water restriction.
SECTION: Taking a Mouse off Water Restriction
26. Allow the mouse to drink it's alloted water amount, and then remove water dish from cage.

 

SECTION: Taking a Mouse off Water Restriction
27. Record on horizontal animal cage card that animal has been placed on adlib water. Include the date.
SECTION: Taking a Mouse off Water Restriction
28. Retrieve the "placed back on adlib" cage flag and place on cage over cage cards. Write the date.
SECTION: Taking a Mouse off Water Restriction
29. Place mouse on non-water restriction rack with water spouts and/or attach a filled water bottle to cage.
SECTION: Taking a Mouse off Water Restriction
29.1. Toggle water spout prior to placing mouse on rack to confirm that water spout is delivering water.
SECTION: Calculating Baseline Weight and Restriction
6. Collect and record daily weight of mouse on ad-lib water for at least 3 days.

 

SECTION: Calculating Baseline Weight and Restriction
6.1. Scoop/cup mouse out of cage.

 

SECTION: Calculating Baseline Weight and Restriction
6.3. Place mouse in weigh container and place on scale. Note the weight.
SECTION: Calculating Baseline Weight and Restriction
7.1. Toggle water spout prior to placing mouse on rack to confirm that water spout is delivering water.
SECTION: Calculating Baseline Weight and Restriction
6.4. Return mouse back to cage. 

 

SECTION: Calculating Baseline Weight and Restriction
6.5. Clean weigh container with 70% Ethanol spray after each use. This is to prevent any additional stress on mice caused by previous mice excrement.

 

SECTION: Calculating Baseline Weight and Restriction
9. If Waterlog is not accessible, manually calculate the mouse's baseline and target weight.

SECTION: Calculating Baseline Weight and Restriction
9.2. Multiply your baseline weight by the desired water restriction percentage (as specified by your IACUC protocol). 

For example: 
- Mouse has a baseline weight of 25.00 grams.
- Desired water restriction at 85% of weight.
- 25.00 grams x 0.85 = 21.25 grams.
- Maintain mouse at target weight of 21.25 grams. 

 


SECTION: Calculating Baseline Weight and Restriction
11.1. Record mouse information on on vertical water restriction card including:
- Trainer name
- Watering time (AM or PM)
- Mouse ID
- Workflow
- Baseline weight

Record daily mouse watering information including:
- Date
- Time
- Total water provided
- Initials

 

SECTION: Calculating Baseline Weight and Restriction
11.2. Record that animal has been placed on water restriction on horizontal animal cage card. Include your initials and the date placed on water restriction. 

 

SECTION: Calculating Baseline Weight and Restriction
12. Return mouse to water restriction rack (racks with detached water valves) in AHR.
SECTION: Calculating Baseline Weight and Restriction
1. Open Waterlog via desktop application.
SECTION: Calculating Baseline Weight and Restriction
3. Enter mouse ID in mouse ID text box and press enter on your keyboard.

 

 


SECTION: Calculating Baseline Weight and Restriction
2. Enter your LIMS/SLIMS User ID.

 

SECTION: Calculating Baseline Weight and Restriction
6.6. Record mouse weight in weight textbox of Waterlog. Click submit button.
SECTION: Calculating Baseline Weight and Restriction
7. Place mouse on non-water restriction rack with water spouts and/or attach a filled water bottle to cage for the first two days.
SECTION: Calculating Baseline Weight and Restriction
4. Retrieve mouse from the animal holding room (AHR).
SECTION: Calculating Baseline Weight and Restriction
5. Ensure that mouse has correct enrichment in their cage based on requirements of the experimental design and genotype (i.e. tube, wheel, chewing block, etc.).
SECTION: Calculating Baseline Weight and Restriction
8. On the third day use Waterlog to calculate the average of your recorded weights to obtain the mouse's baseline weight. 

 

SECTION: Calculating Baseline Weight and Restriction
8.2. Enter mouse weight into the current weight text box. Enter any behaviorally relevant information (behavior program title, animal performance notes, hardware/software issues, etc.) in the comment text box.

 

SECTION: Calculating Baseline Weight and Restriction
8.1. Open Waterlog and enter LIMS User ID.
SECTION: Calculating Baseline Weight and Restriction
8.3. Place mouse on water restriction within waterlog.

 

SECTION: Calculating Baseline Weight and Restriction
8.4. Click the "Water Restrict and Define Baseline Weight" button (outlined in green above).
SECTION: Calculating Baseline Weight and Restriction
8.5. Confirm that the calculated baseline weight appears to be correct (average weight over 3 days).

 

SECTION: Calculating Baseline Weight and Restriction
8.6. Confirm mouse ID, sex, pedigree and protocol between mouse horizontal cage card, "links" dropdown menu in Waterlog, and personal schedule. 
Confirm baseline weight, target ratio, and target weight calculations in Mouse Details section of Waterlog page.

 

 

SECTION: Calculating Baseline Weight and Restriction
10. Provide mouse with suggested water amount. 

 

SECTION: Calculating Baseline Weight and Restriction
10.1. Using the water bottle with syringe, pull up 1.0mL of water and provide to mouse via water dish in cage.

 

 

 

SECTION: Calculating Baseline Weight and Restriction
11. Record necessary information on physical cards on mouse cage.
SECTION: Calculating Baseline Weight and Restriction
8.9. Obtain a water dish.

 

SECTION: Calculating Baseline Weight and Restriction
8.8. Prepare a bottle with rack water and a 1mL syringe.
 
 

 


SECTION: Maintaining a Mouse on Water Restriction
16. Confirm mouse ID, sex, pedigree and protocol between mouse horizontal cage card, "links" dropdown menu, and personal schedule. 
Confirm baseline weight, target ratio, and target weight calculations in Mouse Details section of Waterlog
 


SECTION: Maintaining a Mouse on Water Restriction
14. Retrieve mouse cage from AHR. Confirm mouse ID on all cage cards.
SECTION: Maintaining a Mouse on Water Restriction
20. If Waterlog is not accessible, manually calculate the mouse's water allotment.
SECTION: Maintaining a Mouse on Water Restriction
20.1. Water allotment calculation (no previous water rewards):
- Mouse weight is 20.00 grams.
- Baseline weight is 21.25 grams.
- 21.25 grams - 20.00 grams = 1.25 grams.
- 1.0mL = 1.0 grams.
- Provide mouse 1.25 mL of water.

Water allotment calculation (previous water rewards):
- Mouse weight is 20.00 grams.
- Baseline weight is 21.25 grams.
- Mouse received 0.75mL of water reward.
- 21.25 grams - 20.00 grams = 1.25 grams.
- 1.0mL = 1.0 grams.
- 1.25 mL - 0.75mL = 0.5mL.
- Provide mouse 0.5 mL of water.

 

 

SECTION: Calculating Baseline Weight and Restriction
8.7. Within the AHR, locate the hose for the external cage rack water supply. This is typically behind the cage racks.

 

SECTION: Calculating Baseline Weight and Restriction
9.1. Calculate the average weight of the mouse with a minimum of three recently collected weights. This is your baseline weight. 
SECTION: Calculating Baseline Weight and Restriction
10.2. Select the "delivered" checkbox and click the "submit" button to the right to submit the mouses watering information for the day. 
SECTION: Maintaining a Mouse on Water Restriction
13. For each day the mouse is on water restriction, the mouse will need to be given their calculated water atollment each day. 
SECTION: Maintaining a Mouse on Water Restriction
17. Weigh mouse for each day on water restriction to maintain mouse at target weight.

 

SECTION: Maintaining a Mouse on Water Restriction
17.1. Scoop/cup mouse out of cage.

 

SECTION: Maintaining a Mouse on Water Restriction
17.3. Place mouse in weigh container and place on scale. Note the weight.
SECTION: Maintaining a Mouse on Water Restriction
17.4. Return mouse back to cage. 

 

SECTION: Maintaining a Mouse on Water Restriction
17.5. Clean weigh container with 70% Ethanol spray after each use. This is to prevent any additional stress on mice caused by previous mice excrement.

 

SECTION: Maintaining a Mouse on Water Restriction
17.6. Record mouse weight in weight textbox of Waterlog. 
SECTION: Maintaining a Mouse on Water Restriction
17.2. Check mouse for correct ID (ear notches and/or tattoo). Check mouse for any other wellness concerns (headframe, prolapse, wounds, etc.).
SECTION: Calculating Baseline Weight and Restriction
6.2. Check mouse for correct ID (ear notches and/or tattoo). Check mouse for any other wellness concerns (headframe, prolapse, wounds, etc.).
SECTION: Maintaining a Mouse on Water Restriction
18. Enter water amount received prior to weighing in "already earned" textbox.
SECTION: Maintaining a Mouse on Water Restriction
19. Provide mouse with [now] calculated water allotment as written in "suggested" textbox.

 

SECTION: Maintaining a Mouse on Water Restriction
21. Record mouse watering information on vertical water restriction card including:
- Date
- Time
- Total water provided
- Initials
SECTION: Maintaining a Mouse on Water Restriction
22. Return mouse to water restriction rack (racks with detached water valves) in AHR.
SECTION: Taking a Mouse off Water Restriction
30. Place used water dish in dirty bin of AHR.
SECTION: Calculating Baseline Weight and Restriction
4.1. If transporting mice under reverse light cycle, utilize a cart cover within non-red light rooms.