Additives to the Drinking Water for Rats and Mice (IACUC)

Last updated on May 8, 2023 5 min read Additives to the Drinking Water for Rats and Mice (IACUC)

It is often convenient as well as less interruptive to the animals to add medication and experimental compounds to the drinking water, rather than administering them by gavage or parenterally. A critical factor to be determined when any compound is added to the drinking water is the animals’ fluid intake. It must be assured that fluid intake will not be diminished and the animals will not become dehydrated because of any new taste, smell, texture, or other factor altering the drinking water. This policy outlines procedures to be implemented for any and all drinking water additives.


  1. Any compound added to the drinking water must be approved by IACUC protocol.
  2. Ad lib water consumption must be determined before any compound is added to the drinking water.
  3. Compounds that are commonly used and with which the principal investigator (PI) is familiar and can provide data or references that the water intake will not be reduced are exempted from pre-determination of ad lib water consumption. These include, but are not limited to, sulfatrim (trimethoprim-sulfamethoxazole). For compounds that have not been previously administered and for which data are not available, the PI must provide data as described in the Procedures section below. BU ASC will attempt to maintain a database allowing the information of tested additives to drinking water to be shared between investigators.
  4. If animals will receive a choice of unadulterated drinking water in addition to a water bottle with additives, as in a preference study, making a choice of drinking water possible, the study is exempted from pre-determination of ad lib water consumption.
  5. Monitoring for hydration by evaluating skin turgor, food consumption, and body weight must be done for novel compounds and documented daily for a minimum of three days. Animals should be weighed at least twice weekly during the first week.
  6. The PI is responsible for labeling all drinking water with additives.
  7. A compound such as sucrose may be added to the drinking water in order to make the experimental compound or medication more palatable (see Antibiotics Added to the Drinking Water in the Procedures section, below).


Evaluation of Ad Lib Water Consumption

  1. Prior to introducing a new compound in the drinking water, the PI must determine the strain’s typical 24-hour water consumption.
  2. The mean daily (24-hour) water consumption varies between species, stocks, and strains. It is therefore mandatory to determine the ad lib water intake for the same strain, sex, age, and weight rodents as will be used in the study. As an example, the mean daily water intake in mice has been reported averaging 7.7 +/- 0.3 ml/30g body weight and varying 2-fold across strains.1
  3. Once the ad lib water consumption has been determined for one strain of mice or rats or other rodent species, ad lib water intake need not be repeated.
  4. Published data, or data from the planned Boston University Animal Science Center (BU ASC) database, if available, may be used.

Method for Determining 24-hour Ad Lib Water Consumption

    1. Rodents are housed in their usual cage.
    2. A Special Care Instruction card stating “water is being measured DO NOT CHANGE WATER BOTTLE” is placed on the cage to prevent animal care staff from changing the water bottle or interfering with the study.
    3. The water bottle is filled (~200ml–400ml) and weighed using a digital scale to 0.1g accuracy.
    4. The same sipper tube the animals are accustomed to is used for the study.
    5. The bottle with water is weighed 3 days in a row at the same time of day to determine 24-hour ad lib water consumption.
    6. The three measurements are averaged and then divided by the number of rodents in the cage.
    7. Alternative methods may considered as reviewed and approved by the IACUC.

    Adding the Novel Compound to the Drinking Water and Documentation of Fluid Intake

    1. Volume of the water with additive consumed must be measured each day, as described above, and documented over a period of at least three days.
    2. When a novel compound is added to the water, daily observations of the rodents to evaluate hydration and fluid and food consumption must be documented for the first three days.
    3. If rodents drink less than 90% of their previous intake, become dehydrated, or lose weight, the PI must adjust the drinking water with additives so as to make it palatable for the animal. If the problem persists, the PI must consult with a BU ASC veterinarian.

    Antibiotics Added to the Drinking Water

    1. Sulfamethoxazole/Trimethoprim (Sulfatrim)
      • Stock solution: 200mg Sulfamethoxazole +40mg Trimethoprim/5ml
      • MOUSE: Dose 95mg/kg/24 hours, 5.3ml of stock in 400ml drinking water2
      • RAT: Dose 53mg/kg/24 hours, 4.4ml of stock in 400ml drinking water2
      • Suspension: Shake bottle daily
      • Light sensitive: Cover bottle with foil or dispense in colored bottle
    2. Doxycycline (as antibiotic)*
      • MOUSE: Dose 5–10mg/kg/24 hours, 5.3ml of stock in 400ml drinking water
      • RAT: Dose 5–10mg/kg/24 hours, 4.4ml of stock in 400ml drinking water
      • Suspension: Yes
      • Light sensitive: Yes
      • Doxycycline is bitter and consequently it is advisable to add 2.0–5.0% sucrose to the water
      • *Doxycycline is also used as a genetic modifier (dose to be determined by the PI)
    3. Tetracycline
      • MOUSE: 50–60mg in liter drinking water
      • Tetracycline is bitter and consequently it is advisable to add 2.0–5.0% sucrose to the water

    Both Sulfamethoxazole-Trimethoprim and Enrofloxacin were assessed for stability in drinking water and found to be stable up to 7 days. Plasma concentrations of the drugs were notably lower when administered by mouth.3 To support immunosuppressed strains, investigators should consider whether operational exclusion of pathogens (barrier IVC housing, sterile caging, irradiated feed, Hydropac® chlorinated water) is adequate without administration of antibiotics in the drinking water. However, in some cases, use of antibiotics in drinking water may still be required to limit morbidity and mortality (e.g. in the short term after irradiation).


    1. Bachmanov, AA, Reed, DR, Beauchamp, GK and Tordoff, MG. Food intake, water intake, and drinking spout preference of 28 mouse strains. Behav. Genet. 2002. 32:435–443. For detailed information, please see the Mouse Phenome Database.
    2. Hawk, C. Terrence Eds. Formulary for Laboratory Animals. Third Ed. 2005. Blackwell Publishing. Rat and mouse doses calculated from allometric correction from large animal dose (30mg/kg/day).
    3. Marx,JO, Daljit,V, Murphy, L, Rankin, S, Hankenson, FC. Antibiotic Administration in the Drinking Water of Mice. J Am Assoc Lab Anim Sci. 2014 May; 53(3): 301-36.

    BU IACUC approved May 2011, Revised January 2014, Revised August 2018, Approved September 2018, Revised and Approved October 2021

    Information For...

    Back to Top