Rodent Fluid Restriction

Introduction

Boston University is committed to observe Federal Guidelines and AAALAC International Guidelines for Humane Care and Use of Animals.

This policy deals with fluid regulation as part of the experimental protocol. Experimental reasons for introducing fluid regulation fall into three main categories, as follows: 1) studies of homeostatic regulation of energy metabolism or fluid balance, 2) studies of the motivated behaviors and physiologic mediators of hunger or thirst and 3) studies that regulate fluid consumption to motivate animals to perform novel or learned tasks.

It is recommended that Principal Investigators consult with a University Veterinarian prior to submitting a protocol involving fluid restriction or regulation to the IACUC for review.

Food and Fluid Monitoring Form

I. Definition of Fluid Regulation1

A. Scheduling: Scheduling access to fluid sources so an animal consumes as much as desired (ad lib) but only at regular intervals.

B. Restriction: The total volume of fluid consumed is strictly monitored or controlled and animals will not receive their complete daily ad lib ration.

II. Policy

A. PI is required to consult with veterinary staff before starting a fluid regulation study.

B. Animals on fluid regulation or restriction must be monitored daily and findings documented daily using hyperlinked form. Animals on fluid restriction must be weighed at least twice a week at least 48 hours apart.

C. Rodents on fluid regulation or fluid restriction must be identified by a card on their cage See procedures for LASC vs LACF P. 4, VII.A. and B.

D. Scheduling of fluid intake: animals are given ad lib access to water at least:

  1. Once every 24 hours for one hour for rats and
  2. Once every 12 hours for one hour for mice

E. Animals must be acclimated gradually, over a period of at least 3 days, to new restriction/regulation paradigms.

F. Fluid restriction must be limited to a carefully calculated minimum daily volume required to keep the animal healthy.

G. Before a fluid restriction study is started, it is mandatory that the ad lib daily fluid intake is determined for the exact same strain, sex and age to evaluate their normal, ad lib fluid intake. Specifics for this requirement are detailed on P.3, V.

H. “For both types of fluid regulation (scheduling vs. restriction), animals generally should be given free access to fluid for some period on days when experimental sessions are not scheduled, unless scientifically justifiable reasons preclude such fluid supplementation.”1

I. Planned duration of fluid regulation/restriction must be specified in the IACUC protocol.

J. No rodent can be completely deprived of fluids for more than 24 hours.

K. Research staff responsible for monitoring animals on fluid regulation studies must be trained and competent to evaluate the animal’s condition.

L. “In the case of conditioned-response research protocols, use of a highly preferred fluid as positive reinforcement, instead of restriction, is recommened”.5

M. The investigator must use “the least regulation/restriction that will achieve the scientific objective.”5

N. Young or growing animals are especially sensitive to fluid restriction and placing these classes of animals on fluid regulation/restriction must be evaluated with a concern for their health and minimum growth requirements.

O. Fluid regulation for research purposes must be scientifically justified, and a literature search for alternatives must be performed.

P. Supportive Care and Interventional Endpoints must be observed as specified in this Policy, Section VI, or, if different, specified in the IACUC protocol.

Q. Depending upon the severity of the fluid regulation/restriction paradigm, the IACUC will consider assigning the animals to USDA Pain and Distress Category E2.

R. In case of LASC/LACF finding a dehydrated animal every effort will be made to contact the assigned research staff and Principal Investigator. However, if it is not possible to contact these individuals, the animal will be treated according to directions from the Attending Veterinarian or designee.

Procedures

III. Consideration of the species’ circadian rhythm, behavior and physiology.

A. Rodents, being nocturnal, eat and drink primarily at night during the dark period. Therefore, removing water in the evening for overnight restriction basically results in a restriction lasting 24 hours, since they have probably not drunk much during the previous day.

B. The influence of the circadian rhythm on eating and drinking behavior can only be physiologically adjusted ~10% (2-3 hours) per 24 hours.8 Thus it may take 4 days or longer for the animal to become acclimatized to a change in the timing of provision of fluids.

C. “Efforts should be made to match an animal’s typical watering schedule within circadian variables.”1

D. Animals fed without accessible water or fed and watered out of schedule will eat less and lose weight.

E. Determination of the Minimum Fluid Requirement of each animal is required in fluid restriction studies. Minimum fluid requirements are indirectly evaluated as state of hydration. Enough fluids must be provided on a daily basis to keep the animal from becoming dehydrated.

F. “The physiologic effects of water deprivation are critically dependent on composition of the maintenance diet … with up to 25% daily fluid requirement required for metabolism of the typical rodent high fiber chow.”8

IV. Species-specific Considerations: Mice

A. Mice, being one logarithmic unit smaller in body weight than rats, have correspondingly higher Basal Metabolic Rate (BMR) and “correspondingly faster water turnover”.8

B. Mice therefore are less tolerant of water restriction and PIs must be aware of extending observations made on rats to mice6,8.

C. Considerable strain variation in mean daily water intake has been reported in mice averaging 7.7 +/- 0.3 ml/30 g body weight and varying 2-fold across strains.9

D. It is therefore extremely important to establish mean daily water intake for the particular strain to be used in each study.

V. Mandatory Determination of ad lib fluid intake prior to fluid restriction study –Procedures

A. Ad lib fluid intake must be determined for the same strain, sex, age and weight rodents as will be used for the study. There are significant variations in fluid intake based on these parameters. Once the ad lib fluid consumption has been determined for one strain of mice, ad lib fluid intake must not be repeated. Published values for the same strain, sex, age and weight may be used in lieu of in-house determination. If published data are not available, ad lib fluid intake must be determined by one of the methods described below.

B. Method used by Bachmanov, A.A.9
Mice are housed individually in plastic shoe box cages with bedding and covered with a S/S grid lid (N = 6-8). Water is supplied via a graduated drinking tube. “Each drinking tube consisted of a 25 ml plastic serological pipette with 0.2 ml gradations (Fisher Cat. No. 13-678-14B). This was connected to a 63.5 mm long stainless steel sipper tube (Unifab, Cat. No. US-171=25) with a 15 mm piece of silicon tubing (Cole Palmer, Cat.No.06441-76). The top of the pipette was closed with a size 00 rubber stopper. The drinking tubes were placed in the mouse’s food hopper. The spouts were extended 25 mm into the mouse cage.”9 Repeat measurements 3 consecutive days is required with the 3 data points averaged to determine ad lib 24 hours fluid intake.. Measurements must be taken every 24 hours at a set time of day.

C. Alternative method to be used for either rats or mice
Rodents are housed individually in plastic shoe box cages with bedding. A water bottle, is filled with water (~200 ml) and weighed using a digital scale to the 0.1 g accuracy. The same sipper tube as the animal is used to is used for the study. 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. The 3 measurements are averaged.

D. The effect of food on fluid intake
The kind of food an animal eats affects fluid consumption. Therefore, the food given during the study must be the same type as that fed during the determination of ad lib fluid intake. Ingestion of rodent chows requires the animals to drink enough to compensate for the fiber and other components in the chow. Physiologic needs for water are influenced by many factors, including the water and electrolyte content of the diet, the ambient temperature and humidity, and exercise. Fluid consumption is also influenced by non-hydration variables, such as habit, social factors, palatability, and ease of access to fluids.”1

VI. Supportive Care and Interventional Endpoints– Fluid Restriction

A. Dehydrated animals must be treated immediately by supplying a measured volume of water for drinking. In addition, if the animal is recumbent or more than 5% dehydrated, SC Lactated Ringer’s Solution (LRS) must be administered. Supplemental oral fluids or parenteral hydration must be administered whenever any of the following clinical signs are observed:

1. Dehydration of > 5% defined as “clinical dehydration”. This is evaluated as loss of skin turgor (tenting of skin) in rodents.
2. No urine output for > 12 hours. Bedding is dry in rodent cages. There are few or no feces. Feces are dry.
3. Urine specific gravity is increased above normal. It may be possible to get a drop of urine on a dip stick by picking up the rodent and holding it over a Petri dish or a urine dip stick. Alternatively, in order to evaluate urine specific gravity, it may be necessary to house the rodent in a metabolic cage and collect urine.
4. Lack of appetite and body weight loss of more than 10% (2.5 g in a 25 g mouse or 25 g in a 250 g rat) over <48 hours. Dehydrated animals will not eat and will limit their food intake.
5. Note that dehydration will also cause weight loss. A 5% dehydrated animal will have lost 5% or more of its body weight.
6. The eyes appear sunken.
7. The animal is listless and inactive.
8. An animal that has been fluid restricted for some time must be given supplemental water in carefully graded portions, in order to prevent over-hydration with subsequent detrimental physiologic consequences.

VII. Special card on cage required for rodents on food regulation or restriction

A. LASC PI or research staff are responsible for identifying rodents on fluid regulation or fluid restriction by placing a yellow “Special Care Instructions” card on their cage.

B. LACF
PI or research staff are responsible for identifying rodents on fluid regulation or fluid restriction or any other special care by submitting a a request form to LACF Director. This form is then laminated by LACF and posted on the door of the animal room. Colored dots are used to indicated water regulation (blue dot) or food regulation (red dot). Detailed feed or watering instructions are included on the special care request form. LACF is available to water holidays and weekends.

VIII. RODENTS – SCHEDULING OF FLUID INTAKE

A. Removing water from rats or mice may be done in the morning, in preparation for a fluid reward activity later in the day such as maze learning or pedal pressing for a fluid reward. If fluids are removed in the evening, the actual period of fluid restriction may be closer to 24 hours rather than 12-16 hours.

B. After the study session is completed, the rodent is returned to the home cage with ad lib food and water.

C. The session is documented on the hyperlinked form and body weight must be checked and documented at least twice a week at intervals of at least 48 hours.

D. When using fluid rewards as motivation for task performance, it is imperative for the investigator to ensure that the daily requirements to maintain a healthy state are met by the sum of earned rewards and supplemental fluid offered.
E. It is recommended that the animals be allowed an opportunity to work for extra rewards until satiated, even if additional data in not needed, or be given free access to make-up water to meet the minimum daily requirement.

IX. RODENTS – RESTRICTION OF FLUID INTAKE

These studies may be designed as:
A. “Animal is given access to a measured volume of fluid per day and may consume that volume over any length of time.”1

B. Animal is given free access to fluids for a defined period of time.

C. The estimated daily fluid maintenance requirement in small rodents is 100 ml/kg/24 hours. This amounts to 2.5 ml for a 25 g mouse and 25 ml in a 250 g rat. Note that the actual ad lib measured fluid intake in several mouse strains is much higher.9 Species and strain variations are commonly seen. Therefore, it is incumbent upon any investigator planning to apply fluid restriction for longer than 24 hours in research rodents (=chronic restriction), to determine 24 hour ad lib fluid intake for the particular strain, age, sex and body weight as described on P.3, V. Then, the study animal is allowed a calculated percentage of the ad lib consumption. Enough fluids must be administered to replace daily losses as evaluated by the animal’s hydration status, as discussed in VI. Food should be provided concurrently with fluids as the animal benefits from simultaneous availability of food and fluid.

D. “For long-term fluid restriction, the animal must be gradually weaned down over a period of 3 days to the consumption goal. In addition, once the long-term restriction protocol is no longer required, animals must be brought back to ad libitum gradually over a period of 3 days. During this time they should be monitored closely for deleterious effects of fluid overload (i.e., gastrointestinal complications like bloat or neurological signs).”3

E. For one time, acute fluid restriction lasting < 24 hours which may be relevant to certain studies, PI is required to describe and justify this in the IACUC protocol and documentation using hyperlinked form.
LASC
PI or research staff are responsible for identifying rodents on fluid regulation or fluid restriction by placing a yellow “Special Care Instructions” card on their cage.
LACF
PI or research staff are responsible for identifying rodents on fluid restriction or any other special care by submitting a request form to LACF Director. This form is then laminated by LACF and posted on the door of the animal room. Colored dots are used to indicated water regulation (blue dot) or food regulation (red dot). Detailed feed or watering instructions are included on the special care request form. LACF is available to water holidays and weekends.

References

1. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. ILAR. NRC. 2002. Food and Fluid Regulation. PP. 49-61.
2. APHIS Policy 11. Painful procedures.
3. MIT CAC Guidelines on Food and Water restriction in Rodents, 2007.
4. University of Michigan UCUCA. Policy on Food and Water Restriction or Manipulation in Laboratory Animals. 2007.
5. The Guide for the Care and Use of Laboratory Animals. ILAR. NRC. 1996. P.12.
6. Schmidt-Nielsen, Knut. Scaling. Why is Animal Size so Important? Cambridge University Press.1984.
7. Toth, Linda A. and Gardiner, Thomas W. Food and Water Restriction Protocols: Physiological and Behavioral Considerations. Contemp. Topics 39(6), Nov. 2000
8. Rowland, Neil E. Food or Fluid Restriction in Common Laboratory Animals: Balancing Welfare Considerations with Scientific Inquiry. Comparative Medicine, 57(2), April 2007. (Highly recommended).
9. 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

http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=meas/catlister&req=Cmetabolismqqqwater%20intake

BU IACUC Approved March 2009