Food Regulation and Restriction in Rodents

Introduction

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

This policy deals with food regulation and restriction as part of the experimental protocol.  It does not address food restriction to animals to maintain optimal, healthy body weight, and it does not address fasting prior to anesthesia and surgery.  Neither does it address experimental diets which do not entail caloric restriction, for example high cholesterol diets.  These topics are addressed elsewhere in the appropriate IACUC policy.

Experimental reasons for introducing food regulation or restriction fall into four main categories, as follows: 1) studies of homeostatic regulation of energy metabolism, 2) studies of the motivated behaviors and physiologic mediators of hunger, 3) studies that regulate food consumption to motivate animals to perform novel or learned tasks and 4) studies that regulate food consumption to study the effect of caloric restriction on disease processes such as aging or cancer.

Food and Fluid Restriction Monitoring Form

I. Definitions

A. Food regulation: Food regulation includes food scheduling and food restriction.1

B. Scheduling: Scheduling access to food sources so an animal consumes a full serving but only at regular intervals.1 No change in weight occurs relative to age-matched control animals.

C. Restriction: The total volume of food consumed is strictly monitored or controlled.1 The total calories fed to and consumed by the animal is strictly monitored and controlled to reduce the weight of the animal to a level below that of age-matched, full-fed control animals.

D. Full-fed: Most nonrodent animals are fed a ration in the usual laboratory setting. The amount fed to animals NOT on food-regulation studies is designated “full-fed” or “full meal.” Full-fed animals have a Body Condition Score (BCS) of about 3 (=normal; not too fat and not too thin). If they are young and growing they will gain weight on this ration.

E. Ad lib fed: Only rodents are routinely fed ad lib in the usual laboratory setting. Ad lib is defined as “The animal is allowed more food than it consumes and can eat as much as it wants whenever it wants”.

F. Every-other-day (EOD) feeding, also called “intermittent feeding” is a accepted feeding regimen used in caloric restriction and aging studies in rodents. Rodents are fed ad lib EOD for 24 hours. They are fasted for the following 24 hours. Their body weight may be >20% less than ad-lib fed, age-matched controls and they have longer life spans. 11, 12, 13

II. Policy

A. Consultation with a University Veterinarian prior to submitting a protocol involving food restriction or regulation to the IACUC for review is required and will expedite the approval process.

B. Animals on food regulation or restriction must be monitored daily and findings documented using the hyperlinked form. The form must be kept in the animal room for LASC/LACF information.

C. Rodents on food regulation or food restriction must be identified by a card on their cage See procedures for LASC vs LACF P. 3, VI.A. and B.

D. Severe food restriction studies must not be started until rodents are at least 14 weeks of age10.

E. The goal for body weight loss must be limited to the animal reaching > than 80% of an age- and sex-matched ad lib fed control unless scientifically justified in the IACUC protocol.

F. Planned duration of food regulation/restriction must be specified in the IACUC protocol.

G. Rodents completely deprived of food for more than 24 hours will express certain physiological and behavioral adaptations which may be necessary for certain studies. Complete food deprivation of 72 hours in rats and 48 hours in mice is acceptable with scientific justification8.

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

I. “In the case of conditioned-response research protocols, use of a highly preferred food as positive reinforcement, instead of restriction, is recommended”.5 However, exceptions to this recommendation or alternatives may be allowable if scientifically justified.

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

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

L. Food restriction studies must assure that the diet is nutritionally adequate so that the animal’s metabolic requirements are met and the animal receives the minimal daily requirement of protein, fats, carbohydrates plus vitamins and minerals to stay healthy. Above applies unless the study investigates the minimal nutritional requirement of a certain food component or if the intent of the study is to test hypotheses related to the pathophysiological effects of nutrient deficiency or weight loss.

M. Food regulation for research purposes must be scientifically justified in the IACUC protocol, and a literature search for alternatives must be performed.

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

O. Depending upon the severity of the food regulation paradigm, the IACUC will consider assigning the animals to USDA Pain and Distress Category E2.

P. In case of LASC/LACF finding an animal on food regulation in distress 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. “Efforts should be made to match an animal’s typical eating schedule with circadian variables.”1

B. Rodents, being nocturnal, eat and drink primarily at night (dark cycle), interspersed with other activities. Therefore, removing the food in the evening for overnight restriction basically results in a restriction lasting 24 hours, since they have probably not eaten much during the previous day.

C. Species-specific eating habits should be considered.

D. Determination of the Minimum Caloric Requirement to maintain health of each animal is required in food restriction studies.

IV. Supportive Care and Interventional Endpoints – Food restriction:

A. Supplemental food must be administered whenever the following clinical signs are observed:

1. No fecal output for > 24 hours. There are few or no fecal pellets.
2. Body weight loss of > 10 % in 7 days (one week) or > 20% in > two (2) weeks.
3. Rodents on food restriction studies must be weighed 3X/week during the acclimation period; then at least weekly unless the animal’s condition warrants more frequent body weight monitoring.
4. After the animal has reached 80% of the body weight of an age- and sex-matched control the daily food allowed should be enough to keep it at this weight and not lose more weight.
5. Body condition score < 2
6. The animal is listless and inactive.

V. Acute up to 24 hours one-time fasting

For a one time, acute food restriction (=fasting) lasting < 24 hours which may be relevant to certain studies, such as fasting for blood glucose (BG) determination or metabolic testing, PI is required to describe and justify this in the IACUC protocol. In addition, documentation using hyperlinked form and a card (See VI, A or B) on the rodent cage is required to alert animal care staff to the temporary removal of food.

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

A. LASC PI or research staff are responsible for identifying rodents on food regulation or food restriction by placing a yellow “Special Care Instructions” card on their cage.B. LACF PI or research staff are responsible for identifying rodents on food regulation or food 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 feed holidays and weekends.

VII. RODENTS – SCHEDULING OF FOOD INTAKE

A. Rodents are given ad lib access to food at least once every 24 hours for at least one hour.

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 attached hyperlinked form and body weight is checked and documented weekly. During the first 1-2 weeks of a food scheduling study the animals must be weighed and the weight documented three (3) times per week.

VIII. RODENTS – RESTRICTION OF FOOD INTAKE

Restriction of food (total daily caloric intake)
These studies may be designed as:

A. Feeding the animal a percentage (50 – 70%)of ad lib consumption daily to reach a body weight of no less than 80% of ad lib fed controls.

B. Reducing an animal’s body weight by up to 20 % compared with an ad-lib fed, age-matched control1.

C. The feeding is documented on the attached hyperlinked form. During the first 1-2 weeks of a food scheduling study the animals must be weighed and the weight documented three (3) times per week. BCS is determined and documented weekly.

IX.GUIDELINES FOR CALCULATING FOOD RESTRICTION AND CALORIC INTAKE

A. When using food restriction as a method of training and/or motivation for task performance, the animal’s strain, age, weight, and life stage must be considered. For example, certain strains (i.e., Zucker Rat) are prone to obesity while younger animals have additional requirements for growth.

B. As a guideline the average 25g adult mouse will consume ~15g/100g BW/day of a nutritionally balanced diet containing 12-14% protein and 4-5% fat9. Thus mice will eat ~3.75g diet/day. PMI 5001, a commonly used rodent diet, has 3.02 Kcal/gm. Hence 3.75 g would provide 11.3 Kcal. Adult mice will eat 4-5 g pelleted ration daily. Some of the larger strains may eat as much as 8 g per day per animal.9 Using allometric scaling formulas6, a mouse size mammal (0.03 kg) would require 168 kcal/kg/24 hours = 5.05 kcal to maintain Basal Metabolic Rate (BMR) at rest. To maintain some daily activity, the caloric requirement would be higher as indicated above.

C. The average 300g adult rat will consume ~5g/100 g BW of a nutritionally balanced diet containing 5-14% protein and 4-6% fat9. Thus adult rats will consume ~15 g diet/day. Fifteen (15) g PMI 5001 would provide 45.3 Kcal. “Rats will eat varying amounts depending upon their genetic origin. Larger strains will eat 15 – 30 g per day. Smaller strains will eat 12 -15 g per day”.9 Using allometric scaling formulas6, a rat size mammal (0.3 kg) would require 94.6 kcal/kg/24 hours = 28.4 kcal to maintain Basal Metabolic Rate (BMR) at rest. To maintain some daily activity, the caloric requirement would be higher as indicated above.

D. If young growing animals (rats or mice <14 weeks of age)10 are to be restricted, the investigator must ensure adequate nutrient intake to avoid malnutrition. The use of non-feed restricted littermates for comparison is optimal, but otherwise vendor growth chart comparisons for the appropriate strain can be used. Body weight in growing animals must be no less than 80% of an age-, sex, and strain-matched control animal.

E. The investigator must ensure that the nutritional value of the earned and free food is sufficient to maintain the animal’s health status. The use of nutritionally balanced food treats (raisins, peanuts, etc.) is encouraged. However, if other treats such as fruit loops are used, the animal should only be offered the amount needed to complete the session. For food restriction protocols, animal(s) should be gradually weaned down to the desired body weight or consumption goal. LASC/LACF recommend BIO-SERV http://www.bio-serv.com/
for nutritionally balanced treats.

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. P.12. 1996.
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. Purina Mills Lab Diets. http://www.labdiet.com/
10. National Institute of Aging: Guidelines for Caloric Restriction in Rodents
11. Goodrick, CL et.al. 1982. Effects of intermittent feeding upon growth and life span in rats. Gerontology 28(4):233-241.
12. Goodrick, CL et.al. 1983. Effects of intermittent feeding upon growth, activity, and lifespan in rats allowed voluntary exercise. Exp. Aging. Res. Fall 9(3):203-209.
13. Goodrick, CL et.al. 1990. Effects of intermittent feeding upon body weight and lifespan in inbred mice: interaction of genotype and age. Mech Ageing Dev. July 55(1):69-87.

BU IACUC Approved March 2009