September
October 2012

Volume 5
Number 5

Dosing Animals Via Diet
This can be a simple and efficient method for delivering
compounds to lab animals when done correctly.
Matthew Ricci, PhDO

ften, experimental protocols call for dosing animals
with a compound.Traditional dosing methods such asoral gavage or injections have significant drawbacks.
One important consideration is the stress associatedwith handling the animal.The animal’s stress response
can affect their behaviour and metabolism, potentiallyconfounding the parameters you are measuring. Many
animal handlers will admit that such dosing can also be
stressful for them as well.The other key issue is the time
involved in these methods of dosing. Even an experienced
person would likely take about 2 hours to gavage 100
mice—time which can be used for other purposes.
The idea of dosing animals by adding a compoundto their diet has been around for quite some time andis successfully used with everything from experimental
drugs to approved pharmaceuticals to food/plant extracts.
When dosing via diet, the concerns of both handlingrelated stress and the time involved are removed.
So what should be taken into consideration whendeciding how to dose animals via diet?
The researcher must first realise that oral administration(whether via diet or gavage) will almost surely produce
different pharmacokinetics (what the body does to a drug)
compared to an injection.This is because when taken
orally, the liver and intestine have a chance to metabolise
the drug before it reaches the general circulation.
Injections bypass the liver and intestine and so wouldbe expected to result in different pharmacokinetics.
Furthermore, a single daily dose given via oral gavage
and the same dose consumed in the diet over the courseof the animal’s nocturnal feeding are likely to produce
different plasma levels of the compound.This may result indifferent effects on the biological systems being measured.
Also, the researcher should determine whether any

dietary ingredients have the potential to interfere with theabsorption of the compound.This is likely to be more of apotential issue when using grain-based diets as comparedto purified ingredient diets (see below).
Once the researcher decides to dose the compound viadiet, there are other decisions to make.
What type of diet should I use?
Lab animal diets fall into two main categories: purified
ingredient diets or grain-based ‘chow’ diets. In a previous
issue of ALN World’s sister publication, ALN (September/October 2005), we discussed in detail the differences between
these types of diets. Briefly, chow diets, while completely
nutritionally adequate, often contain non-nutritive substancesthat can affect the phenotype of the animal. Examples include
phytoestrogens (which can have pro- or anti-estrogenicactivity) and heavy metals, such as arsenic. Some researchers
have had eye-opening experiences when they discovered thatthe estrogen or arsenic they were giving to the animal (eitherorally or via injection) had little effect on the animal precisely
because they had already and unknowingly ‘dosed’ the animal
via diet. Because the content of the relatively unrefined plantmaterial used in chow diets can change over time and acrossharvest locations, there can be variation from batch to batchof chow diets.And, because each ingredient in chow diets
contains multiple nutrient (and non-nutrient) materials, it isdifficult to fine-tune the content of a chow diet.An example
would be removing some or all of a specific vitamin from thediet—this is really not possible using a chow diet.
Purified ingredient diets, as the name implies, usehighly refined materials, with the goal that each ingredientessentially adds only one nutrient.The advantages ofusing purified ingredients include minimal batch tobatch variability, absence of extraneous materials such as

feature | Drug Dosing
phytoestrogens or heavy metals and ease of formula revision.
If a researcher decides to add a compound to a purified
ingredient diet for animals previously fed a chow, theyshould first switch the animals to the new diet (withoutadded compound) for at least a few weeks for acclimationand to ‘wash out’ any effects of the prior diet.This periodof time will depend on the parameters being measured(plasma cholesterol, gene expression, etc.). Since chowsand purified diets are different in many ways, feeding a
new diet can change the phenotype.Therefore, it would
not be recommended to simultaneously change theirdiet and dose them with a compound—the effects of thecompound could be clouded by the diet change itself.
Is the compound stable to diet production
conditions?
If the researcher is concerned that their compound will
be destroyed during diet production, they should ask thediet manufacturer about their methods. If the researcher
wants powdered diet, compound stability should be less of aconcern, since the ingredients plus the compound are simply
mixed together without further manipulation (see later
comments on using powdered vs. pelleted diet). If pelleted
diet is desired, the water added (required for pelleting) andheat generated during pelleting are conditions that theresearcher should know about, as they can potentially affectthe stability of the compound.To enter some animal facilities,
the diet needs to be irradiated and so the potential effects of
gamma irradiation on the compound should be discussed.
Can the compound be homogenously
incorporated into diet?
Most of the time, the answer is yes and the dietmanufacturer should be able to tell you this based on thephysical characteristics of your compound.What physicalform is the compound? The best type to work with is a freeflowing powder, since it will easily mix well with the otherpowdered diet ingredients. If the compound is a hard crystal,
it would first need to be ground into smaller, finer particles
and this can be done either by the researcher or the dietmanufacturer. Sometimes, the compound has a syrup-likeconsistency. If it is soluble in ethanol, the resulting solution
can be poured into the diet, since the ethanol evaporates
fairly quickly, leaving the compound evenly distributed
throughout the diet. Problematic compounds include thosewith very high water content, such as slurries—especially
if the researcher wants the compound added at a relatively
high dose. High water content in lab animal diets can leadto microbial growth and inability to form a pellet, and so inthese cases it would be best if the compound were first dried
down to a powder.This is most often done by the researcher
or a commercial freeze-drying facility.
Should I feed the diet as a pellet or a powder?
Most researchers prefer pelleted diet for several
reasons—it is easier to feed, easier to measure food intake,less diet is wasted and there is no chance that ingredients
will ‘settle out’ and no longer be uniformly distributed
throughout the diet.There is even the chance than animals
could avoid compound rich areas in powdered diet. One
reason for using powdered diets is that the researcher
has the freedom to mix in the compound themselves(perhaps trying many different doses in a pilot study). In
these situations, the researcher will need to adequately
mix the diet plus compound.This is more difficult for theresearcher to do in the case of high fat diets. High fat diet‘powders’ have a consistency of something like sticky, wetsand and so the researcher should be aware that it may
not be possible to homogenously mix in their compoundat this stage. (Ideally, the compound would be mixed withthe dry ingredients and a small amount of the fat earlier
in the mixing process). Finally, if the compound is notstable to pelleting conditions (addition of water and mild
heating), using powdered diet would be necessary.
How can I calculate the diet dose?
In order to determine how much compound needs to beadded to the diet, you will need to know 1) the daily dose
you want to achieve (per kg body weight), such as 3 mg/kg,2) the animal’s body weight and 3) the animal’s daily foodintake. In the equation below, DD is the diet dose (in mg/kg
diet), SD is single, daily dose you want to deliver (in mg/kg),
BW is body weight (in g) and FI is daily food intake (in g).
DD = (SD X BW)/FI
This obviously works best if you have an accurate ideaof your animals’ weight and how much they eat. Of course,
growing animals will weigh more over time, but theywill also eat more, and the researcher should continue
to monitor these parameters in order to continuously
calculate the dose over time.
A brief word here about dosing multiple diets that havedifferent caloric densities.A good example of this is highand low fat diets, which contain a different number ofcalories per gram of food given that fat is more caloricallydense than carbohydrate and protein.When a researcher
wants to add a compound to both a high and low fat diet,they will need to decide whether to add the compoundto the diets on a weight or calorie basis.A weight basis
means that the compound would be added at say, 100 mg/kg in each diet.A calorie basis means that the compound
would be added at say, 100 mg/4,000 calories in diet.Thisdecision is important because very often animals will ‘eat
for calories’, not for weight. For example, rats and mice willoften eat fewer grams of a high fat diet when switched froma low fat diet because high fat diets contain more calories
per gram of food than low fat diets.There is usually a periodof adjustment (a week or two) after which the low and highfat fed groups eat the same number of calories of food (butnot the same number of grams). If the diets were dosedwith compound based on weight, the high and low fat
groups would be receiving different amounts of compoundeach day. If they are dosed based on
calories, the assumption is made thatthe groups will eat the same numberof calories, on average, over time
and therefore eat the same dose ofcompound.
Do I feed the diet ad libitum?
The above discussions (calculating
the dose, dosing by weight or bycalories) assumes that the animals
will be given the diet ad libitum,
which is the most common practice.
That is, they will dose themselvesin the normal course of eating
food when they choose to eat it.
This method clearly reduces theinvolvement of humans, since weonly need to offer the animals plentyof food and let them eat normally.
Another, less common method
would be to deliver the compound ina discrete amount of diet, somethingmuch less than their normal average
food intake (metered feeding). If forexample, a researcher wanted theanimal to eat all of the compoundin a relatively short amount of time,they could offer only one pellet (ora fraction of a pellet) containing thefull day’s dose of compound. Since
rodents are mainly nocturnal eaters, if

they were offered this small amountof diet toward the end of the lightcycle, they would likely consume it as
they should be preparing to become
more active and eat. Another way to
improve the chances that the animal
will eat all of the pellet would be to
first fast them for a time, increasing
their drive to eat when presented
with food. The drawback to thesemethods is that unless you watchthem consume this small amount offood, you cannot be sure that they
have eaten all of it, when you wantthem to eat it. Also, a fasted animal
will have a different hormonal stateand metabolism, and this itself could
potentially change the effects of thecompound. So while metered feeding
does still have advantages over morestressful procedures like oral gavage
or injection, it does require morework than pure ad libitum feeding.

this will change the dose the animals
receive. However, if the dose in thediet is relatively small, it is unlikely tohave any measurable effect on foodintake from a palatability perspective.
In addition, unless the compound is
toxic, the animals will typically eat
the diet since it is their only source offood. Of course, it may be a good idea
to do a pilot study to test this, thoughmore often than not, this is not an
area of great concern.
In summary, dosing via diet is anestablished and simple method fordelivering a compound to lab animals.
It greatly reduces stress and saves
time over other procedures such asoral gavage and injections. Finally,
as long as you have accurate bodyweight and food intake data, youcan easily calculate the dose they
are receiving over the course of anexperiment.

Will the compound affectthe palatability of the diet?
This is often a difficult question
to answer with certainty since
defining palatability in lab animals is
another area of study altogether. If thecompound itself gives the diet a flavor
or taste that changes food intake,

Matthew R. Ricci, PhD is VicePresident/Science Director for
Research Diets, Inc.; +1 732-2472390 x1041; Ricci@ResearchDiets.
com. Research Diets, Inc., 20 Jules
Lane, New Brunswick, NJ 08901
USA; www.ResearchDiets.com.

Posted from ALN World, September/October 2012. Copyright © Vicon Business Media. All rights reserved.
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Dosing Animals Via Diet- Page 4.pdf

1

11/29/12

1:15 PMSimple, Safe Dosing
Research Diets, Inc. will incorporate your compounds into any experimental diet. Feeding test
compounds eliminates dosing related stress to the animal, eliminates vehicle effects, and saves
time and labor. Consult with one of our scientists on the formula, determine the dosage
required and the diet will be produced and shipped in 5 to 7 business days.
Consistent OpenSource Diet® formulation provides a clean, repeatable control diet for yourresearch. Precise, graded addition of test compounds to your specified control diet allows
evaluation of dose response effects in your animal model. We can blend your compound
homogeneously into any diet, typically to ppm and even as low as parts per billion.

Typical FormulaC

Product #Protein
Carbohydrate
FatTotal
kcal/gm

D10001gm%
kcal%202166
68
512100.0
3.90

IngredientCaseinDL-Methionine

gm
200
3kcal
800
12Corn StarchSucrose

150
5006002000

Cellulose, BW200

500Corn Oil

50450

Mineral Mix S10001

350Vitamin Mix V10001
Choline Bitartrate

1024001000

3902

M

YCMMYCY

CMYK

Total

info@ResearchDiets.com

Examples of Compounds Added• Chemotherapeutics• COX-2 Inhibitors• Antioxidants• Nutraceuticals• Statins• Insulin sensitizers• NSAIDSYOURCOMPOUND