Vol 96, No 3

May 2009
Pages 292295
Journal of the American Association for Laboratory Animal Science
Copyright 2009
by the American Association for Laboratory Animal Science
292
For the purposes of risk assessment and species extrapolation,
it is preferable to treat laboratory animals with a compound by
the route of administration that would be used in humans. For
many studies, drug delivery necessitates oral administration.
A drug can be administered orally by dissolving it in the drink-
ing water or mixing it into the feed. Other options include oral
gavage and pipetted amounts that are directly consumed by
the animal. In preparation for a study of oral methylphenidate
treatment in rats, we found that each of these techniques had
disadvantages for the focus and endpoints of our study, prompt-
ing us to search for an alternative method.
The most detrimental confounder for our developmental
toxicology laboratory is the potential stress associated with
oral gavage. Gavage is an extremely common method for oral
administration, and daily gavage was used successfully in
our previous studies.
7,8
However, questions have been raised
regarding the potential stress induced by this procedure
3

(but see reference 4 for quantifcation of corticosterone levels
after gavage). Signifcant stress during rodent pregnancy can
alter offspring behavior, endocrinology, neuroanatomy, and
neurochemistry.
1,5,15,19
Finally, where multiple daily adminis-
trations are necessary due to a short half-life of the drug, oral
gavage becomes less practical.
Dietary or water delivery of test substances may avoid the
confounding variable of stress, and these routes are particularly
appropriate for studies in which the substance under investi-
gation is found in food (for example, as used in our studies of
genistein found in soy products
9,10
). However, those routes of
administration are not feasible with drugs or substances that
are unpalatable. Further, the normal fuctuations in daily intake
do not result in administration of an exact dose to each study
subject. In addition, the effects of the drug itself may alter those
normal variations, potentially further decreasing daily doses.
For many drugs, human exposure occurs in bolus, rather than
prolonged or timed-release doses. Because rodents typically
eat and drink over several hours during the dark period,
17,18

dietary drug exposure then occurs over an extended period,
peaking during the dark period.
To minimize stress, some studies implemented pipette ad-
ministration of the drug directly to the animal.
11,12,14
In that
procedure, the volume is collected into a pipette and the rodent
voluntarily consumes the liquid from the pipette tip as it is
dispensed. Like gavage, this method allows administration of
an exact dose. This method works well with individual hous-
ing; however, in social housing conditions, cagemates may
interfere with the administration procedure. The method can
be time-consuming and labor-intensive because it requires that
the technician remain at each cage for the necessary duration
for the animal to fully consume the substance.
For a study of oral methylphenidate treatment in rats, our
laboratory needed an administration method that was time-
effcient, used few personnel, and could be easily accomplished
3 times daily. A literature search revealed studies in which
the drug was placed onto a small cracker or wafer that the
rat then consumed,
2,20,21
but there was little description of
the technical details for this procedure. Here, we describe our
system for administration of methylphenidate 3 times daily
to pair-housed adolescent rats; this system led to little or no
observable change in behavior. Our method is highly accurate
with regard to dose administration and effcient with regard to
time and personnel.
Materials and Methods
We used a semiautomatic precision liquid processor capable
of accurate and precise transfer pipetting (Microlab 500, Ham-
ilton Company, Reno, NV). Two of these systems were in place,
with 1 designated for use with control solutions, and the other
for use with methylphenidate solutions, thereby decreasing the
potential for cross-contamination. Each system was equipped
with a push-button hand-pipettor (Concorde Push-button Hand
Probe, Hamilton Company, Reno, NV) and a 250-L Hamilton
syringe. Each system was interfaced with animal data collection
software developed at the National Center for Toxicological
Research (NCTR) and allowed us to use an automated algo-
rithm to calculate the volumes of solution needed based on
the daily body weight of each rat. A balance (model PM11-K,
MettlerToledo, Columbus, OH) that was interfaced with the
data-collection software was used to weigh the animals.
Wafers (Mini Nilla Wafers, Nabisco, Kraft Foods, Northfeld,
IL) were quartered into approximately equal pieces, which were
placed individually in 30-mL polypropylene cups (Ted Pella,
Use of Food Wafers for Multiple Daily Oral
Treatments in Young Rats
Sherry A Ferguson
*
and Sherin Y Boctor
Many laboratory studies require oral administration of drugs. Dietary administration in food or water is useful, but is not
always the best method. Orogastric gavage can be stressful. Here, we describe in detail a relatively stress-free technique that
can be applied to multiple daily administrations using a palatable food item. This method was successfully used to administer
water or methylphenidate 3 times daily to young pair-housed adolescent rats.
Abbreviations: NCTR, National Center for Toxicological Research; PND, postnatal day.
Received: 18 Sep 2008. Revision requested: 22 Oct 2008. Accepted: 30 Oct 2008.
Division of Neurotoxicology, National Center for Toxicological Research, US Food and
Drug Administration, Jefferson, Arkansas.
*
Corresponding author. Email: Sherry.Ferguson@fda.hhs.gov
293
Food wafers for oral treatment in rodents
were placed in the front and rear of the shelf in close proximity
to the cage. The technician then tapped the cup on the top of
the wire cage lid to gain the attention of the rat and emptied
each wafer piece into the appropriate side of the cage without
removing the cage lid. Dividers were removed approximately
5 min after each treatment time.
Methylphenidate or water treatment. Before the frst treatment
time (that is, 0830) each day, rats were weighed. Those data
were transferred to the NCTR animal data collection software
which calibrated the volume, based on body weight, necessary
for each rat to receive 3 mg/kg of methylphenidate or 1 mL/kg
of water at each of the 3 treatment times. The technician then
placed a wafer piece into each of the 3 polypropylene cups for
the frst rat and, using the hand-pipettor, dispensed an identical
volume onto each of the 3 pieces, 1 for each of the 3 subsequent
treatment times. Each cup then was placed into the tray in the
appropriate position. In the same way, 3 additional wafer pieces
were treated for the second rat in that cage. Figure 3 shows
the room setup and an example of a technician dispensing the
volume onto the wafer pieces.
Redding, CA) each labeled with the cage number and subject
identifcation. Each of 3 aluminum trays (Figure 1) held the
cups for 1 of the 3 daily treatment times; thus, there was a tray
for the 0830 treatment, 1 for the 1130 treatment, and 1 for the
1430 treatment. Trays were confgured for the NCTR cage-rack
system, in which there are 4 shelves per rack, and each shelf
contains 4 cages. Thus, each tray had 4 columns and 8 rows of
cup holders. Because rats were pair-housed, the frst row of 4
cups contained wafer pieces for the frst rat in each of the 4 cages
on the frst shelf. The second row of 4 cups contained wafer
pieces for the second rat in each of the 4 cages on the frst shelf,
and so on, until the eighth row, which contained wafer pieces
for the second rat in each of the 4 cages on the last (fourth) shelf.
Trays were stackable for space-saving purposes.
The usual rat housing cages were modifed slightly by creat-
ing in each cage a slot that was slightly off center. Immediately
prior to each treatment time, a clear acrylic divider (0.32 cm
thick) was placed in each cage to physically separate the 2 rats
in each cage (Figure 2). This divider ft tightly into the cage slot
and could not be moved by the rats.
Animal procedures. All animal procedures followed the Guide
for the Care and Use of Laboratory Animals
13
and were approved
in advance by the NCTR Institutional Animal Care and Use
Committee. Anecdotal observations on a different group of ani-
mals indicated that young rats found cheese crackers (Goldfsh,
Pepperidge Farm, Campbell Foods, Camden, NJ) especially
palatable, but they were not particularly absorbent for the liquid
drug. These observations also indicated that pieces of graham
cracker (Honey Maid, Nabisco), although absorbent, seemed to
be less desirable than the pieces of vanilla wafers (Mini Nilla
Wafer, Nabisco). In preliminary testing of the pipetting systems,
absorbency appeared to be greatest on the back of the wafer
pieces. Therefore, all further procedures used vanilla wafer
pieces. The manufacturers nutritional information indicated
that each full wafer contained 7 calories, or 1.75 calories in each
quarter piece. Thus, each rat received approximately 5.25 ad-
ditional daily calories from this treatment regimen. At no time
were rats food-deprived; all were provided with ad libitum
food [5LOH (pelleted irradiated diet), Purina Mills, St Louis,
MO] and water.
The 3-wk treatment period was postnatal days (PND) 29 to
50, and wafer training began with the arrival of the Sprague
Dawley rats (42 male, 42 female; derived from Crl: COBS CD
BR outbred rats, Charles River, Wilmington, MA) from the
NCTR Breeding Colony at weaning (PND 21). These rats were
pair-housed with a same-sex, same-treatment nonsibling in
standard polycarbonate cages with hardwood chip bedding in
a room maintained on a 12:12-h light:dark cycle (lights, 0700 to
1900) at 22 1 C (mean SEM) and 45% to 55% humidity. On
that day, several (3 to 5) wafer pieces were placed in the cage to
habituate rats to the taste and smell of the wafers. Beginning on
PND 22, body weights were collected each morning, and the 2
rats in each cage were separated (see below) and given untreated
wafer pieces as described below for treated wafer pieces. Dur-
ing these frst few days, technicians observed the rats to ensure
that each piece was consumed entirely. After approximately 3
d, all rats consumed pieces in their entirety within 2 to 3 min
at all 3 treatment times.
To physically separate the rats at each of the 3 treatment
times (0830, 1130, and 1430), the technician placed a divider in
each cage, ensuring that the frst rat (tail tattoo 1 in each cage)
was in the front half of the cage and the second rat (tail tattoo 2
in each cage) was in the rear. The appropriate tray containing
wafer pieces was brought to the rack, and the cups for each cage
Figure 1. A sample tray with cups. This tray is for 1 of the 3 daily treat-
ment times, and the frst 2 rows contain treated wafers for the 4 cages
on the frst shelf (the frst row contains treated wafers for rat 1 in the
cage; the second row contains treated wafers for rat 2 in the cage). Both
the tray and cups are labeled and color-coded for easy identifcation.
The tray measures 45.7 30.5 3.2 cm, and cup holes are 3.2 cm in
diameter.
Figure 2. A typical cage showing the acrylic divider (although clear
when in actual use, an X was placed on the divider for photographic
purposes) in place, with rat 1 in the front of the cage (near the cage
card), and rat 2 in the rear of the cage. The small slot that holds the
divider can be seen.
294
Vol 96, No 3
Journal of the American Association for Laboratory Animal Science
May 2009
This system could easily be implemented with other test
substances or experimental paradigms. For individually housed
rodents, the acrylic dividers would not be necessary. For mice,
the wafers could be divided into smaller pieces, which likely
would still be large enough to accommodate the volume of
methylphenidate used here. The methodology could even be
used for animals housed in wire-bottom cages, because our
observations indicated that each rat held the wafer piece in the
forepaws and consumed it entirely without allowing it to touch
the bottom of the cage. However, our methodology may not be
applicable to all test substances that require oral administration.
For example, compounds with an adverse taste or odor likely
will not be consumed voluntarily, but the adverse taste or odor
could potentially be masked by the addition of a palatable
favor (for example, fruit concentrates) to the solution, such as
the preparations added to childrens medications. This method
may be inappropriate for paradigms in which a large volume
must be administered (for example, due to poor solubility of
the test compound), because the wafer will hold only a limited
volume before becoming soft. Overall, our experience has been
that animal care technicians are enthusiastic about this proce-
dure, there appears to be little observable stress to the animal,
and the experimenters are confdent about the accuracy of the
dose administered.
Acknowledgments
The authors acknowledge Richard Rasmussen (Bionetics Corpora-
tion) for his construction of the trays and cage dividers, Carol Cain
(Bionetics Corporation) for design improvement of the dividers, Clyde
Ulmer and Kathy Carroll (Z-Tech) for creation of the network algorithm,
and Lee McVay (Bionetics Corporation) and the incredibly talented
animal care staff of the Bionetics Corporation at NCTR.
This document has been reviewed in accordance with United States
Food and Drug Administration (FDA) policy and approved for publica-
tion. Approval does not signify that the contents necessarily refect the
position or opinions of the FDA nor does mention of trade names or
commercial products constitute endorsement or recommendation for
use. The fndings and conclusions in this report are those of the authors
and do not necessarily represent the views of the FDA.
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Latency to consume the wafer was measured in subsets of rats
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16
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6
Discussion
A pump system, metal trays with cup holders, and acrylic
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Figure 3. The room setup showing the automated pump system, com-
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hand-pipettor to apply methylphenidate solution to each of the 3 wa-
fer pieces for the 3 daily treatments for a single rat.
295
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