Laboratory Animal Diets:
A Critical Part of Your In Vivo Research
Most all of us are aware that certain dietary choices can increase or decrease the likelihood of developing certain diseases. Our diets can also change our metabolism as well
the levels of circulating factors (hormones, lipids, etc.) which may be markers for diseaserisk. What is often overlooked is the fact that these concepts also apply tolaboratory animals, making diet a critical part of study design.
Matthew R. Ricci, Ph.D. and Edward A. Ulman, Ph.D.

Like humans, the animal’s phenotype, ranging the full spectrum from good health to a disease state, is the resultof the interaction between their genes and their environment. Since the genetic makeup of the animal is fixed,
manipulating their phenotype generally means making subtle or drastic changes in their environment, which
include diet, housing, and ambient temperature. For good and ill, environmental modifications can happen easily and sometimes occur without researchers’ knowledge, leaving them scratching their heads and wondering
why previous results were not repeated under what they thought were identical study conditions. Hence, control over environmental conditions is important to minimising data variability.
Nutritional science research during the 20th century has shown that diet is a powerful environmental toolcapable of changing the phenotype of an animal. Diet-induced disease models rely on diet to drive the desired
phenotype. Examples include diet-induced obesity, diabetes, dyslipidemia, hepatosteatosis, atherosclerosis, andhypertension, to name a few. Diet also plays an extremely important role even when it is not being used purposefully to develop a disease state. For one, diets fed during pregnancy and lactation can have long-term
effects on the phenotype of the offspring. Additionally, diets fed during a toxicology study can affect how thetest compound manifests its toxicological effects. Hence, conclusions drawn about the toxicology of a compound may vary depending on the type of diet fed during the study.

WE ARE ALL NUTRITIONISTS
Given the importance of diet on outcome, how should scientists make choices about what to feed? First, theyshould realise that since they are feeding an animal some type of diet, they should add “nutritional scientist” totheir job description. It is now up to them to embrace this new title (or not). And, as all nutritional scientists
know, it is in their best interest to be involved with and cognizant of the choice of diet fed to their research
animals, as this may save innumerable headaches down the road. Secondly, they should know that while there
is no perfect diet, some have real advantages over others.

REPORT, REPEAT, REVISE
When choosing a diet, one should ask three questions: Can I report it (can I tell others exactly what my animals were fed)? Can I repeat it (is there diet variability and will I be able to get the same results next year)?
Can I revise it (as my hypotheses change, can I easily change the dietary components while keeping it otherwise matched to previous diets)? The answer should be “yes” to all three.

CEREAL BASED DIETS
Laboratory animal diets basically fall into two categories:
chows and purified ingredient diets. Chow diets (Photo 1)
have been used since the 1940s as the “background,” “maintenance” or “control” diet in experiments. They are relatively
inexpensive to produce and provide complete and adequate
nutrition. Referred to as grain or cereal based, these diets typically contain ingredients such as ground corn, ground oats,
alfalfa meal, soybean meal and ground wheat. Vitamins, minerals, and fat are added to ensure nutritional adequacy. Chow formulas are generally “closed” formulas, meaning that the exact
amount of each ingredient added is kept secret by the manufacturer.
An important point to remember is that each of theplant materials in chows contains many compounds, each
inseparable from the next. Some of these are nutritive (protein, carbohydrate, fat, vitamin, minerals, and fibre) and someare non-nutritive (for example, plant derived compounds collectively termed phytochemicals) components. Because thenutritional content of these plant materials will naturally fluctuate with harvest location and across growing seasons, thismeans that the content of chow diets will vary from batch tobatch.
For example, the soybean meal used in a chow today
may not have the same percentage of protein (arguably thenutritional standard by which this ingredient is judged) as
the soybean meal used six months ago. So when making achow, one is left with two choices – to use the same amountof soybean meal every time the chow is made, or to accountfor nutritional differences by adding more of less soybean
meal to “correct” for differences in the protein levels.
Actually, chows are made using both methods and eachhas disadvantages. If soybean meal levels are always kept
constant, then the protein levels of the diet will vary withthe protein levels of the soybean meal. With the second
method, overall protein levels can be roughly maintained byvarying the amount of soybean meal used in a particular
batch of chow.
However, this raises a new issue – in keeping dietary
protein levels constant by changing the level of soybean
meal, what has happened to the levels of non-nutritive components of that soybean meal? Soybean meal (and otherplant-derived ingredients) contains many varied and interesting phytochemicals, numbering in the hundreds. A subclass of phytochemicals is the phytoestrogens. These phytoestrogens can bind to estrogen receptors in the animal andhave either pro- or anti-estrogenic effects. Since the progres-Photo 1: Example of asion of disease states such as“cereal based diet”
obesity, atherosclerosis andcancer can be affected by such
estrogenic or antiestrogenic activity, it may be advisable touse a diet without phytoestrogens altogether. Secondly, ifsoybean meal levels are varied across batches to account fordifferences in protein levels, it follows that the levels of phytoestrogens will vary not only from chow to chow but also
from batch to batch of the same chow. Such variability inphytoestrogens may translate into variability in data overtime, leading to cost increases due to either repeating studies
or having to use to larger numbers of animals per study.
Neither of these outcomes is cost-efficient nor desirable.
Is it easy to report a chow? One can give the name of
the chow being used, but is it really the same as what was fed
last year, especially down to the non-nutritive components?
Arguably, the answer is “no” given the variability in theingredients used. Plus, since most chow formulas are closed,
one can never truly know how much of each ingredient wasused in this particular batch. Is it easy to repeat a chow?
Using the same argument about ingredient variability, theanswer here is also “no”.
Can a chow diet be revised as research hypotheseschange? Revisions can mean removing something from oradding something to a diet. Given that each plant ingredient in chow can contain a dozen (or more) nutrients,removing a nutrient from the chow is not possible. For
example, one could not study the effects of a very low iron
diet chow. There is just no way to remove the iron from anyor all of the plant materials - it is like trying to remove thesugar from a baked apple pie.
This restricts chow revisions to additions. However,there are limitations here as well. As an example, let’s
examine high-fat diets. Given the increasing population ofobese and diabetic people in Westernized cultures, researchin these related areas has increased greatly in the last
decade. Laboratory animals are fed high-fat diets in orderto test the ability of therapeutic compounds to prevent orreverse obesity. While it is possible to make a high fat
chow by mixing fat with powdered chow and either feeding it as such or pelleting the mixture, this should be donefeature|DIETFigure 1: Creating a high-fat chow can dilutethe nutrient concentrations
Ingredient

Chow

Chow (gm)
Lard (gm)
Total

1000
01000

Chow with
20% Fat
800
200
1000

Gram %
Protein
Carbohydrate
Fat

2350
51940
24

Kcal%
Protein
Carbohydrate
FatTotal

2860
12100

1736
48100

with caution, because as fat is added, the nutrient concentrations in the chow are diluted (Figure 1).
In this example, 20% fat has been added to a chow (800gm chow plus 200 gm lard). While this effectively increases
the fat from 12% to 48% of calories, it has also diluted thelevel of protein from 28% to 17% of calories. Thus the protein calories and all other nutritive and non-nutritive components have been reduced by 40%. This can be problematicfor two reasons. First, such overzealous addition of fat candilute the diet enough as to make it protein deficient, clearly
not the intention when studying the effects of a high-fat diet.
Secondly, this dilution effect makes comparisons to the control diet (presumably the unmodified chow) difficult. Not
only will the experimental group be eating a higher fat diet,but they will also be eating less protein, vitamins, minerals
and fiber per calorie of food, relative to the control group.
Hence when comparing data between the groups, it will be
impossible to determine if differences in phenotype were due
to changes in any one nutrient.

“CAFETERIA” DIETS

Figure 2: The AIN-76A Rodent Diet
IngredientgmkcalCasein200800DL-Methionine

312Corn Starch150600

Sucrose500

2000

Cellulose

500Corn Oil

50450

Mineral Mix

350

Vitamin Mix

1040

Choline Bitartrate

2010003902

Total

It is worth spending a short time describing so-called ‘cafeteria’ diets and their shortcomings even though their use isdiminishing due to a greater understanding of the diet choices available to researchers. The first published studies using‘cafeteria’ diets in laboratory animal research appeared justover 30 years ago. As their name implies, these diets offer theanimals a choice of foods found in supermarkets, including
chocolate, nuts, cookies, peanut butter, cheese, salami, toname a few. It is not uncommon for the available food choices to change during the course of the study. To ensure nutritional adequacy, an appropriate laboratory chow diet is usuallyalso offered to the animals.
It should be fairly evident that feeding such cafeteria diets
by definition means that researchers will have very little control over what the animals eat. As a result, it will be impossible to accurately report their nutrient intake. Furthermore,this food self-selection model almost ensures that it will beimpossible to repeat the study, given that another group ofanimals will likely choose a different combination of foods.
Finally, as with chows, it is not possible to make subtle nutritional changes to the diet, since nutrients are being supplied
by many dietary sources and because the animals are making
different food choices every day.
As mentioned earlier, the use of cafeteria diets has thankfully declined in the past decade. However, although mostnutrition-oriented journals do not (and should not) accept
articles that use cafeteria diets, it is still possible to find suchfeature|DIETpapers (interestingly, most recently published papers using
cafeteria diets are from labs in Europe). Twenty years ago,
Dr. Barbara Moore made a convincing argument against
the use of cafeteria diets in a well-written article whose
message remains valid today1.

PURIFIED INGREDIENT DIETS
Purified ingredient diets (Photo 2) were initially used by biochemists and nutritional scientists in their first major, sharedendeavor of delineating the limited list of required nutrients the simplest list of chemicals and molecules required in thediet for life versus death. Later, they studied the interaction
between various nutrients and the influence of diet on more
subtle quality of life (health and disease) issues, like diet andcancer, for example.
The idea behind purified diets is simple: each nutrient is
supplied by a separate, purified ingredient. In the strictest
sense of the terms, purified and semipurified diets differ in thetypes of ingredients used, though today the terms are generallyused to mean the same thing. Purified ingredient diets can be(and we would argue should be) “open source” formulas,meaning that they are published and available to the scientificcommunity. One note of caution on purified diet formulas – if
a researcher is trying to repeat published data, they should makesure that the diet company they are using (if different from theone in the paper) is making the formula exactly as was reported. It is quite possible to find instances in which diets weremade “to be similar to” published formulas, but in reality useddifferent ingredients. Changes in ingredients can lead tochanges in phenotype, something not helpful for the scientist
trying to compare their data to what is in the literature.
In the early days of purified diet use, many research
nutrition groups developed; each using their own favorite
purified diet and usually using making them in house. For
example, Vitamin A researchers developed separate and very
distinct purified diet formulas from those studying Vitamin Dor selenium or Vitamin E. Because of these differences, it
became quite difficult to compare observations across thesenutrient study disciplines, from lab group to lab group. Despite
these differences, the formulas were generally well reported,
allowing one group to know exactly what another group had
fed their animals.
In the early 1970's, the American Institute of Nutrition(AIN) recognized that research nutritionists were travelingdown these many separate tracks and also that other nonnutrition biologists were returning to the fold and using purified ingredient diets to study all aspects of health and disease.
The AIN formed a committee and suggested that a simple

purified ingredient diet be
Photo 2: Example of aadopted for use as a “stanpurified ingredient dietdard” purified diet by all biologists. The result of this collaboration was the AIN-76A rodent diet formula (Figure 2).
In the AIN-76A rodent diet, the protein requirement ismet by the milk protein casein, along with added methionine (to meet sulfur-containing amino acid requirements).
Carbohydrates in this case are supplied by corn starch andsucrose, corn oil provides the fat, and cellulose supplies thefiber. Vitamin and mineral mixes specific to rodents areadded to ensure adequacy. Each nutrient is supplied by aseparate, purified ingredient. It is true that casein, for example contains trace levels of certain vitamins and will containsmall amounts of some minerals. In general, this only
becomes of importance when the goal of the experiment isto induce a deficiency state in one of those vitamins or minerals. In those cases, one can use alcohol-extracted casein(to remove the trace amounts of fat and fat-soluble vitamins) or individual amino acids (the literal links in the protein chain) to lower the background levels of these nutrients.
It is because these ingredients are refined materials,
each containing one nutrient, (as opposed to the less refinedchow ingredients) that allows research nutritionists to define
the nutritional requirements of animals, by selectively
removing one nutrient at a time from the diet. This alsomeans that the possible modifications one can make to a
purified ingredient diet are virtually limitless. This is also
what continues to make purified diets powerful research tools
and why so many scientists have turned to them in recentyears.
First, purified diets are simple to report. For example, apaper may state that “rats were fed the AIN-76A diet for theentire study.” The list of ingredients and their quantities can beeasily and precisely described. Hence, researchers worldwide areable to duplicate the diet should they want to, or compare it to
the diet they are using. And, since there is very little variation
between batches of purified ingredients, the AIN-76A dietmade today will be the same as the AIN-76A diet made a year
from now. This repeatability of purified ingredient diets is clear-feature|DIET

ly advantageous during long-term experiments liketoxicological studies, when variation in data over time
may make interpreting the toxicity of the compound
difficult.

REVISING PURIFIED DIETS
It is in diet modifications where purified ingredient
diets most clearly illustrate their advantage overchow diets. For example, diets with high levels ofsucrose (and no corn starch) have been formulated
and used to study the development of insulin resistance. The fat source can be changed from coconut
oil, to olive oil, to sunflower oil, to study the effects
of changing the fat type from primarily saturated, tomonounsaturated, to polyunsaturated fatty acids,respectively. As mentioned earlier, individual or
multiple vitamins and minerals can be removed to
study their deficiencies and to define requirements.
One key idea here is that when modifications
are made, the remainder of the diet should be identical to the unmodified control diet. This makes
comparisons across experimental groups easy tomake, since only one diet component is changing
at a time. This concept is quite simple to understand when it comes to removing or adding components that do not have caloric content – vitamins
and minerals for example. So when vitamin B6 isremoved from a diet, no calories are removed – justthe vitamin. Hence the experimental and control
diet are different only in presence or absence of thisvitamin.
What about changing dietary components that
contain calories – protein, carbohydrate, and fat?
At this point, it is necessary to introduce a concept
called the nutrient-to-calorie ratio. Not to be confused with the caloric density (the number of calories per gram of diet), this ratio compares the levelof a particular nutrient (or nutrient group) per calorie of diet. Taking another look at the formula for
the AIN-76A rodent diet (Figure 2) we see which
ingredients have caloric content. Using the standard Atwater physiological fuel values of 4, 4, and 9
kilocalories (kcal) per gram for protein, carbohydrate, and fat, respectively, the 500 gm of sucrose,for example, contributes 2000 kcal to the diet. We
now have the information we need to calculate thenutrient to calorie ratio for any nutrient. For example, this diet contains 10 gm of vitamin mix and 50

gm of cellulose per 3902 kcal.
Now that we calculate this ratio, why is it
important? The answer lies in the fact that animalswill for the most part, eat for calories, not weight offood, in an effort to consume the same amount ofcalories over the long term. This means that if ananimal is used to eating a low-fat diet and they areswitched to a higher-fat diet which (because fat issuch an energy-dense nutrient) contains more kcal
per gram of food, they will (after a period of adjustment) spontaneously eat fewer grams of food. They
do this in order to continue eating the same number of calories (not grams) of food as they werewhen eating the low-fat diet. The reverse is true if
switched from a high- to a low-fat diet. Similarly,
rodents will eat more grams of food when the levelsof dietary fiber (which has no caloric content) are
increased, thereby lowering the caloric density ofthe diet. (In reality the ability to eat for calories
does not always hold true – some species/strains will
not regulate feeding and will overeat when exposed
to a very high-fat diet for example).
Knowing that the animals will generally eat for
calories explains why diets of different caloric densities (high- and low-fat diets for example) should
be formulated to have similar nutrient to calorie
ratios. This ensures that per calorie of food consumed (but not per gram), animals consuming dietsof different caloric densities will receive the same
absolute amount of nutrients (except thosechanged by design).
Recall the problem with adding a fat source toa chow diet – the other nutrients were diluted
down as the fat was added. Properly formulated
purified ingredient diets avoid the dilution effectbecause the fat is not added “on top of” the otheringredients but rather replaces carbohydrate. We
could choose to replace protein, but generally thisis not done given the importance of having certain
minimum and adequate levels of protein in thediet.
There are two conceivable ways to replace thecarbohydrate with fat (as an example, see Figure 3).
One way is to switch them on a gram for grambasis, which we argue is the wrong way. In theexample, (using the AIN-76A diet as the startingpoint), 150 extra grams of corn oil were added
while 150 gm of sucrose were removed. However,feature|DIETFigure 3: Replacing Carbohydrates with Fat.
AIN-76A
RODENT DIET

WRONG
Gram for Gram

RIGHTkcal for kcal

since fat is over twice as calorically dense
Ingredientgm% kcal%
gm% kcal%
gm% kcal%as sucrose, this has changed the nutrient
Protein20.3 20.8
20.3 17.5
25.0
20.8
to calorie ratio for the high-fat diet as
Carbohydrate
66.0 67.7
51.0 43.9
39.7
33.1
compared to the low-fat control diet (theFat
5.0 11.5
20.0 38.7
24.6
46.1
AIN-76A). There are 10 gm of vitamin
Total
91.3 100.091.3 100.0
89.3 100.0
mix per 3902 kcal of AIN-76A andkcal/gm
3.90
4.65
4.80
10gm of vitamin mix per 4652 kcal ofIngredient
gm
kcal
gm
kcal
gm
kcal
high-fat diet. Calculating per 3902 kcal
for the high fat diet, this comes to 8.4 gm Casein
200800200
800
200800of vitamin mix per 3902 kcal. So when
DL-Methionine
312312312
the animals of both groups consume theCorn Starch
150
600
150
600
150600same number of calories as we expect
Sucrose500 2000
350 1400
162.5650
they will, the high-fat group will be conCellulose
50
050
0500suming proportionally fewer nutrients(except fat of course) than the low-fat
Corn Oil
50450
200 1800
200 1800
group.
Mineral Mix
350350350
The solution to this is to substituteVitamin Mix
1040
1040
1040
fat for carbohydrate on a calorie-forCholine Bitartrate2020
20calorie basis. Returning to our example,
when we add 150 gm of fat, we are
Total
1000 3902
1000 4652
812.5 3902
adding 1350 kcal, so we should remove
1350 kcal of sucrose (see last panel ofFigure 3). Now, both the high- and low-fat
diets have the same nutrient to calorie ratios - meaning
the ability to report, repeat and revise your diet. Purified
that when both groups consume the same number of calo- ingredient diets can be used to limit data variability due
ries on a daily basis, they will be receiving the sameto diet and to simultaneously induce the desired phenoamount of protein, vitamins, minerals and fiber. Hence,
type. They also provide a clean, consistent background forsuch calorie-for-calorie diet formulation limits the differshort- or long-term studies. Importantly, purified ingredience in the diets to fat and carbohydrate calories, so difent diets are modifiable in just about any way and thus
allow researchers to explore their hypotheses without limferences between the experimental groups can be attribitation. Remember, you are what you eat (with genetic
uted to the varying levels of just these two macronutrients.
contributions in mind) and so are your lab animals.

YOUR ANIMALS ARE WHAT THEY EATWhen it comes to experimental design, it’s important torealise that the diet is not “just the food.” Rather, it’s animportant environmental study component that can andwill affect the phenotype of the animals and therefore thevariability of your data. When publishing their data, scientists should be strongly encouraged to describe theirdiets in the same detail they would use for how they performed a Northern blot or knocked out a gene of interest.
This level of information will only help the reader tocompare their own data or repeat the reported study.
Recognize that if you are doing in vivo research, you are anutritional scientist. While there is no perfect diet, youshould be aware of advantages and limitations of the various diets available. Important to your decision should be

Reference:
1. Moore, B.J. The cafeteria diet - an inappropriate tool
for studies of thermogenesis. J. Nutr. 1987
Feb:117(2):227-31

Matthew R. Ricci, Ph.D. is Vice-President/Science
Director for Research Diets, Inc; ricci@ResearchDiets.com;
www.ResearchDiets.com.
Edward A. Ulman, Ph.D. is President of Research
Diets, Inc.; Ulman@ResearchDiets.com;
www.ResearchDiets.com.