Food and Fluid Regulation and Restriction (IACUC)
Boston University is committed to observation of Federal laws and regulations and AAALAC International accreditation standards for the humane care and use of laboratory animals.
This policy deals with food and fluid regulation and restriction as part of an experimental protocol. It does not address food restriction for weight management or required fasting prior to general anesthesia or sedation. Please see other IACUC policies regarding these topics, as well as the use of special diets. They may also be specified in a particular study in the protocol.
Experimental reasons for fluid regulation or food regulation or restriction fall into four main categories:
- Studies of energy metabolism or fluid balance
- Studies of the motivated behaviors and physiologic mediators of hunger or thirst
- Studies that regulate food or fluid consumption to motivate animals to perform novel or learned tasks
- Studies that regulate food consumption to study the effect of caloric restriction on disease processes such as aging or cancer. This does not apply to fluid restriction.
Associated Forms:
Special Care Request: Animal Care Special Care Request
Monitoring Form: Food and Fluid Restriction Monitoring Form
Please note the nutritional value of the earned and free food must be sufficient to maintain the animal’s health. Treats high in sugar should be limited and only be offered in the amount needed to complete the session. BU ASC recommends BIO-SERV http://www.bio-serv.com/ for nutritionally balanced treats for multiple species.
Definitions
Food regulation: Food regulation includes food scheduling and food restriction.8
Scheduling: Scheduling access to food sources so an animal can feed consume ad libitum but only during scheduled times.8 Weight change is unanticipated with this methodology.
Restriction: The total volume of food or fluid consumed is strictly monitored or controlled.8
Full-fed or Full meal: The amount fed to non-rodent species receiving a ration that are NOT on food-regulation studies is designated “full-fed” or “full meal.” Full-fed animals have a normal body condition score (2.5-3/5). If they are young and growing, they will gain weight on this ration.
Ad libitum fed: Ad libitum is defined as “The animal is allowed more food than it consumes and can eat as much as it wants whenever it wants.” This is standard practice for laboratory rodents.
Every-other-day (EOD) feeding, also called “intermittent feeding” is an accepted feeding regimen used in caloric restriction and aging studies in rodents. Rodents are fed ad lib EOD for 24 hours, then fasted for 24 hours and then the cycle is repeated.
Policies
A. The investigator should use “the least restriction necessary to achieve the scientific objective while maintaining animal well-being.”9
B. Determination of the Minimum Caloric Requirement or Minimum Fluid Required to maintain health of each animal is required prior to starting food or fluid restriction studies.
a. Minimum fluid requirements are indirectly evaluated as state of hydration. Enough fluids must be provided daily to keep the animal from becoming
dehydrated as determined by its appearance, behavior, and physical examination.
b. Ad libitum daily fluid intake should be determined for the same species, strain, sex, and age of the animals to be subjected to this regimen.
c. Regardless of the reason for food restriction, the animal’s strain, age, weight, sex, and life stage must be considered.
d. For food restriction protocols, animal(s) should be gradually (over 1-2 weeks) weaned down to the desired body weight or consumption goal.
C. The IACUC recommends consulting with a BU ASC veterinarian prior to submitting a protocol involving food or fluid restriction or regulation for review as this may expedite the approval process.
D. Food or fluid regulation for research purposes must be scientifically justified in the IACUC protocol, and a literature search for alternatives must be performed.
E. Duration of food or fluid regulation/restriction must be specified in the IACUC protocol.
F. Research staff responsible for monitoring animals on food or fluid regulation studies must be trained and competent to evaluate the animal’s condition.
G. Animals on food or fluid regulation or restriction must be monitored at least once daily and findings documented either cage-side or on a form, such as the one above. All records must be kept in the animal room or anteroom to the animal room.
H. Rodents on food regulation or food restriction must be identified by a card on their cage.
I. Animals must be acclimated gradually, over a period of at least 3 days, to new restriction/regulation paradigms.
J. Animals on fluid restriction must be weighed at least twice a week, at least 48 hours apart.
K. Rodents on food restriction studies must be weighed 3x/week during the acclimation period (1-2 weeks), and at least weekly unless the animal’s condition warrants more frequent monitoring
L. Even on feed or water regulation, minimum requirements for adequate nutrition and hydration should be met. Exceptions for scientific reasons should be justified and clear endpoints should be detailed in the study protocol.
M. The goal for body weight loss should be limited to the animal reaching no less than 80% of an age- and sex-matched ad libitum-fed control unless scientifically justified in the IACUC protocol.
N. Protocol endpoints for fluid restriction or regulation should detail appropriate loss in body weight or condition as compared to age-matched controls of the same species, strain, age, and sex.
O. Supportive Care and Interventional Endpoints must be observed as specified below in the Procedures section of this Policy or, if different, specified in the IACUC protocol.
P. Maximum restrictive intervals
a. Rodents deprived of food for more than 24 hours will express certain negative physiological and behavioral adaptations, which may be necessary for certain studies or could interfere with others. Complete food deprivation of 72 hours in rats and 48 hours in mice may be acceptable with scientific justification, but is not without risk.11, 13, 14
b. No rodent can be completely deprived of fluids for more than 24 hours.
Q. Scheduling of fluid intake2, 6: Animals are given ad libitum or measured access to water at least once every 24 hours.
R. “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
S. Considerations for young and growing animals
a. Severe food restriction studies (>10% restricted) should not be started until rodents are at least 14 weeks of age.7
b. Young or growing animals are especially sensitive to food and fluid restriction, and placing these classes of animals on food restriction must be evaluated with a concern for their health and minimum growth requirements.
T. “In the case of conditioned-response research protocols, use of a highly preferred food or fluid as positive reinforcement, instead of restriction, is recommended.” Exceptions to this recommendation or alternatives may be allowable if scientifically justified.5
U. Depending upon the severity of the food or fluid regulation paradigm, the IACUC will consider assigning the animals to USDA Pain and Distress Category E.16
V. In the case of BU ASC finding an animal on food or fluid regulation in distress every effort will be made to contact the assigned research staff and Principal Investigator. If it is not possible to contact these individuals, the animal will be treated according to directions from the Attending Veterinarian or designee. Such treatment may include immediate provision of food or water, parenteral nutrition or fluids, or both, if the animal appears able to survive.
Cage Card Requirements by Campus for Food or Water Regulation or Restriction
A. BU ASC – MED: 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. BU ASC – CRC: PI or research staff are responsible for identifying rodents on food regulation or food restriction or any other special care by submitting a Special Care Request form to BU ASC (CRC) office (link above). This form is then laminated by BU ASC and posted on the door of the animal room. Colored dots are used to indicate water regulation (blue dot) or food regulation (red dot). Detailed feed or watering instructions are included on the Special Care Request form. BU ASC is available to feed holidays and weekends.
Procedures
Consideration of Circadian Rhythm, Behavior, and Physiology
- “Efforts should be made to match an animal’s typical eating schedule with circadian variables.”8
- Removing food or water for rodent overnight restriction may result in longer restriction and more severe consequences than intended due to their nocturnal behavior.
- The influence of the circadian rhythm on eating and drinking behavior can only be physiologically adjusted ~10% (2–3 hours) per 24 hours.11 Thus, it may take 4 days or longer for the animal to become acclimatized to a change in the timing of the provision of fluids.
- Species-specific eating and drinking habits should be considered.
- Animals fed or watered out of schedule will eat less and lose weight.
- “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.”11
Rodents — Scheduling, Restricting Food or Fluid Intake
Scheduling food intake
- Rodents are given ad libitum access to food at least once every 24 hours for at least one hour.
- After the study session is completed, the rodent is returned to the home cage with ad libitum food and water.
- The session is documented on the Monitoring Form, and body weights are taken in accordance with the IACUC protocol or this Policy.
Restriction of food (total daily caloric intake)
These studies may be designed as:
- Feeding the animal a percentage (50–70%) of ad libitum consumption daily to reach a body weight of no less than 80% of ad-libitum-fed controls.
- Reducing an animal’s body weight to no less than 80% of an ad-libitum-fed, age-matched control.8
- The feeding is documented on the Monitoring Form, and weighing is performed in accordance with the IACUC protocol or this Policy.
- If young growing animals (rats or mice less than 14 weeks of age7) 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, however 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 unless scientifically justified.
As a guideline, the average 25 g adult mouse will consume ~15 g/100 g BW/day of a nutritionally balanced diet containing 12–14% protein and 4–5% fat.10 Adult mice will normally eat 4–5 g of pelleted ration daily. The average 300 g adult rat will consume ~5 g/100 g BW of a nutritionally balanced diet containing 5–14% protein and 4–6% fat.10 “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.”10
Restriction of fluid of Rodents
Mice have higher Basal Metabolic Rate (BMR) and “correspondingly faster water turnover.”
1 Mice therefore are less tolerant of water restriction and PIs must be aware of extending observations made on rats to mice.
11, 12 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 twofold across strains.
1 It is therefore extremely important to establish mean daily water intake for the particular strain to be used in each study. 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 libitum measured fluid intake in several mouse strains is much higher.
1 Species and strain variations are commonly seen.
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 libitum fluid intake for the particular strain, age, sex and body weight as described in Mandatory Determination of Ad Libitum Fluid Intake section. Then, the study animal is allowed a calculated percentage of the ad libitum consumption. Enough fluids must be administered to replace daily losses as evaluated by the animal’s hydration status, as discussed in Supportive Care and Interventional Endpoints. Food should be provided concurrently with fluids as the animal benefits from simultaneous availability of food and fluid.
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.
For one-time fluid restriction lasting less than 24 hours, the PI is required to describe and justify this in the IACUC protocol and document using the Monitoring Form.
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.
Mandatory Determination of Ad Lib Fluid Intake Prior to Fluid Restriction
A. Ad libitum 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. 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 libitum fluid intake is to be determined by one of the methods described below or via a similar approach. Food also impacts hydration status and enough fluid must be consumed to compensate for the dry matter in the rodent chow. Other factors include the water and electrolyte composition of the diet, ambient temperature and humidity, exercise, palatability of fluid, behavioral/social factors, and fluid accessibility.
8B. Method used by A. A. Bachmanov1
Mice are housed individually in plastic shoe box cages with bedding and covered with a stainless steel grid lid (n = 2-5 animals). 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.”1 Repeat measurements on three (3) consecutive days is required with the three data points averaged to determine ad libitum 24-hour fluid intake. Measurements must be taken every 24 hours at the same time of day.
C. Alternative method to be used for rodents
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 0.1 g accuracy. The same sipper tube as the animal uses to is used for the study. The bottle with water is weighed three days in a row at the same time of day and the mean of the weights calculated to determine 24-hour ad libitum water consumption.
Scheduling Fluid Intake of Rodents
Removing water from rats or mice may be done in the morning, in preparation for a fluid reward activity later in the day. If fluids are removed in the evening, the actual period of fluid restriction may be closer to 24 hours as mentioned previously. After the study session is completed, the rodent is returned to the home cage with ad libitum food and water. The session is documented on the Monitoring Form and body weight must be checked and documented at least twice a week at intervals of at least 48 hours.
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 through the fluid reward and supplemental fluid offered. It is recommended that the animals be allowed an opportunity to work for extra rewards until satiated, even if additional data is not needed, or be given free access to make-up water to meet the minimum daily requirement.
Supportive Care and Interventional Endpoints
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, Lactated Ringer’s Solution (LRS), physiologic saline (0.9% NaCl), or other approved fluid support must be administered via subcutaneous injection. Restorative fluid administration (orally, subcutaneously, and/or intraperitoneally) must be administered whenever any of the following clinical signs are observed:
- Dehydration of greater than 5% defined as “clinical dehydration”, determined by loss of skin turgor (tenting of skin) in rodents.
- Urine specific gravity (USG) is increased above normal. Testing USG may not be reasonable in small rodents, but 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. However, no urine may be obtainable if the rodent is severely dehydrated.
- No urine output for greater than 12 hours. Bedding is dry in rodent cages. There are few or no feces. Feces are dry.
- 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 less than 48 hours. Dehydrated animals will not eat and will limit their food intake.
- The eyes appear sunken.
- The animal is listless and inactive and hair coat ruffled rather than smooth.
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.
Increased food/caloric supplementation should be initiated in food restriction studies if the following clinical signs are noted:
- If the body condition score of the animal falls below a 2/5.
- If the animal’s body weight falls below 80% of an age-, sex-, and strain-matched control animal unless previously justified in the protocol.
- The animal is listless and inactive and hair coat ruffled rather than smooth.
If at any point, any of these clinical signs are noted in an animal, please contact animal care staff immediately.
References
- Bachmanov AA, Reed DR, Beauchamp GK., Tordoff MG. (2002). Food intake, water intake, and drinking spout side preference of 28 mouse strains. Behavior genetics, 32(6), 435–443. https://doi.org/10.1023/a:1020884312053
- Bekkevold CM, Robertson KL, Reinhard MK, Battles AH, Rowland NE. (2013). Dehydration parameters and standards for laboratory mice. Journal of the American Association for Laboratory Animal Science: JAALAS, 52(3), 233–239.
- Goodrick CL, Ingram DK, Reynolds MA, Freeman JR, Cider NL. (1990). Effects of intermittent feeding upon body weight and lifespan in inbred mice: Interaction of genotype and age. Mech Ageing Dev 55(1): 69-87. https://doi.org/10.1016/0047-6374(90)90107-Q
- Goodrick CL, Ingram DK, Reynolds MA, Freeman JR, Cider NL. (1983). Effects of intermittent feeding upon growth, activity, and lifespan in rats allowed voluntary exercise. Exp Aging Res. Fall;9(3):203-9. doi: 10.1080/03610738308258453. PMID: 6641783.
- Goodrick CL, Ingram DK, Reynolds MA, Freeman JR, Cider NL. (1982). Effects of intermittent feeding upon growth and life span in rats. Gerontology. 28(4):233-41. doi: 10.1159/000212538. PMID: 7117847.
- Guo ZV., Hires SA, Li N, O’Connor DH, Komiyama T, Ophir E, Huber D, Bonardi C, Morandell K, Gutnisky D, Peron S, Xu NL, Cox J, Svoboda K. (2014). Procedures for behavioral experiments in head-fixed mice. PloS one, 9(2), e88678. https://doi.org/10.1371/journal.pone.0088678
- National Institute of Aging. Caloric Restricted Colony. (Accessed November 2021) https://www.nia.nih.gov/research/dab/eligibility-criteria-use-nia-aged-rodent-
colonies/caloric-restricted-colony - National Research Council. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Institute for Laboratory Animal Research. Washington, DC: National Academies Press, 2002. Food and Fluid Regulation. 49–61.
- National Research Council. Guide for the Care and Use of Laboratory Animals, 8th edition. Institute for Laboratory Animal Research. Washington, DC: National Academies Press, 2011, p. 31.
- Purina Mills Lab Diets. http://www.labdiet.com/
- Rowland NE. (2007). Food or fluid restriction in common laboratory animals: balancing welfare considerations with scientific inquiry. Comp Med. Apr;57(2):149-60. PMID: 17536615.
- Schmidt-Nielsen, K. Scaling: Why Is Animal Size So Important? Cambridge: Cambridge University Press, 1984.
- Swoap SJ, Gutilla MJ. (2009). Cardiovascular changes during daily torpor in the laboratory mouse. American journal of physiology. Regulatory, integrative and comparative physiology, 297(3), R769–R774. https://doi.org/10.1152/ajpregu.00131.2009
- Swoap SJ. (2008). The pharmacology and molecular mechanisms underlying temperature regulation and torpor. Biochemical pharmacology, 76(7), 817–824. https://doi.org/10.1016/j.bcp.2008.06.017
- Toth LA, Gardiner TW. (2000). Food and water restriction protocols: physiological and behavioral considerations. Contemp Top Lab Anim Sci. Nov;39(6):9-17. PMID: 11487246.
- United States Department of Agriculture, Animal and Plant Health Inspection Service, Policy 11. Painful/Distressful Procedures April 14, 1997. https://www.ncbi.nlm.nih.gov/books/NBK99537/
- University of Michigan ULAM. Guidelines on Experimental Food or Water Restriction or Manipulation in Laboratory Animals. 2017. (Accessed November 2021) https://az.research.umich.edu/animalcare/guidelines/guidelines-experimental-food-or-water-restriction-or-manipulation-laboratory
History
Effective Date: 02/04/2025
Next Review Date: 02/03/2028