Available literature on rabbit nutrition offers conflicting advice on feeding our laboratory lagomorphs.

1
What the hay? Does feeding an alfalfa based diet
predispose laboratory rabbits to hypercalcemia
and renal disease? Eden Paster, DVM, Deb
Hickman, DVM, MS, DACLAM, VA Medical Center,
Portland, OR
Abstract
Hypothesis
Results
Discussion
Data Serum Calcium levels Monthly serum
calcium levels for test animals ranged from 12.5
to 15.3 mg/dl and all remained within normal
range throughout the study (normal range 8.0
15.5 mg/dl). Serum calcium levels did not vary
significantly between timothy and alfalfa fed
groups on average per month or cumulatively
(Table 2), nor did they vary significantly once
the rabbits had been on the new timothy diet for
six months (evaluated and compared at the last
data point) (Table 3).
Feeding alfalfa or timothy diets does not appear
to alter serum or urine calcium levels, or renal
function and structure, over a six month period.
There was no significant difference between
cumulative serum calcium or final serum calcium
measurements after six months. This was
surprising, considering that the difference in
dietary calcium levels are almost two-fold.
However, if the rabbits are able to regulate
their serum calcium through healthy homeostasis,
proper parathyroid function, and secretion
through urine, serum calcium levels would not
have wide variability. What was interesting was
the difference in serum calcium levels at the
first data point, when animals were initially
started on their test diet. At the Oregon
Rabbitry rabbits are fed Purina 5321. We
expected the serum levels to be lower in timothy
fed rabbits, but instead they were higher. This
is unexpected, but could be explained if the
rabbits, after acclimating to the Purina diet ,
had a period of serum calcium irregularity
because they were switched to a diet lower in
calcium. If their bodies were responding to a
lower dietary calcium, there may have been a
compensatory temporary serum calcium release from
tissues until homeostasis was re-established. We
will be investigating this finding further by
continuing our study and taking more frequent
data points at the time of initial diet
change. Urinary calcium levels did not differ
significantly at any time points. Although the
means of the two treatment groups differed by
almost 20, there was so much variability in
measurements for each animal that urine calcium
did not appear a consistent measurement. This
was surprising, considering that literature
suggests rabbits have urine calcium levels that
vary with serum calcium levels and that excess
calcium is excreted in the urine. A complicating
factor to these measurements is that serum and
urine calcium measurements were not always done
concurrently (urine was sometimes collected later
in the day or next day), at the same time of day,
or at the same time interval following feeding.
All of these factors may affect transient changes
in serum and urine calcium levels that would not
be captured by single time points. Other factors
to be considered in this study are rabbit age and
gender. All of the rabbits in this study were
female and 6 months old. Because calcium
requirements are higher in younger animals (0.4
for growth) they may tolerate, and even benefit,
from the higher calcium and protein levels in
alfalfa diets. This age was chosen for our study
because of the applicability to rabbit age seen
in the research environment however, in adult
rabbits maintenance calcium requirements
decrease. In studies where rabbits are allowed
to age or in the pet trade, adult and geriatric
rabbits may not be as tolerant of high dietary
calcium. If maintained on alfalfa diets
long-term, we may also see pathologic renal
changes. Gender may also be a factor that could
be further studied.
Available literature on rabbit nutrition offers
conflicting advice on feeding our laboratory
lagomorphs.  Historically, acceptable rabbit
diets have consisted primarily of pellets with an
alfalfa base.  Suggested diets for pet rabbit
diets have evolved to become primarily hay based
with pellet supplementation, based on rabbit
digestive physiology.  Exotic animal
veterinarians now recommend low protein, high
fiber diets consisting primarily of timothy hay
and timothy based pellets, rather than alfalfa. 
An evaluation of the available commercial diets
for laboratory rabbits found that all pellets,
even the high fiber varieties, are alfalfa
based.  Because alfalfa is higher in calcium than
timothy, alfalfa based pellets contain 1.2
calcium, which is higher than even the published
lactational requirement of rabbits (0.75). 
Timothy pellets, on the other hand, contain about
half the calcium of alfalfa based diets.  In
addition, although hypercalcemia is known to
cause renal disease in other species, research is
lacking in examining effects of increased dietary
calcium on serum calcium levels and resultant
physiologic consequences in rabbits.  Literature
suggests that lagomorph serum calcium levels are
not as closely regulated as in other mammalian
species, and fluctuate in direct proportion to
dietary calcium.  Calcium is also excreted in the
urine in direct proportion to dietary calcium. 
Our hypothesis was that rabbits fed an alfalfa
based pelleted diet would have increased serum
calcium resulting in increased renal pathology
compared to those rabbits fed a timothy based
pelleted diet.  One group of rabbits was fed a
high fiber, alfalfa based diet (control) while
another group was fed a commercially available
timothy based diet that is not currently used for
laboratory species.  Comparisons of serum calcium
levels and urine calcium revealed slightly
elevated serum calcium values for the
experimental group in comparison with the control
group, but still within the reported normal
range.  Urine calcium in the alfalfa group was
almost three times that of the timothy group.  
Our data suggests that the timothy based diet
does not lower serum calcium but does increase
urine calcium levels and, therefore, excretion.
Our hypothesis was that rabbits fed an alfalfa
based pelleted diet would have increased serum
calcium and urine calcium, resulting in increased
renal pathology compared to those rabbits fed a
timothy based pelleted diet.
Materials and Methods
Diets
Table 2 means and standard errors of cumulative
monthly serum calcium levels (mg/dl).
Alfalfa Timothy
Mean 13.7 14.5
St Dev 0.2 0.2
Purina Rabbit Chow (Alfalfa) LabDiet Rabbit HF (Alfalfa, high fiber) Oxbow Bunnybasics/T (Timothy)
Protein gt 16 14.5 gt 14
Fat gt 1.5 2.2 - 3.3 gt 1.5
Fiber gt 17, lt 20 21.1 gt 25, lt 29
Calcium gt 0.6, lt 1.1 1.2 gt 0.35, lt 0.85
Phosphorus gt 0.4 gt 0.25
Salt gt 0.5, lt1 gt 0.2, lt 0.7 gt 0.5, lt 1.0
Alfalfa Timothy
Mean 14.1 14.6
St Dev 0.2 0.4
Table 3 means and standard errors of serum
calcium levels (mg/dl) for test groups at final
(6 month) data point.
However, at the first time data point (initial
measurement of serum calcium levels when animals
are switched from diet received at Western Oregon
Rabbitry to the test diet at month 1), there was
a significant difference between serum calcium
levels of rabbits fed alfalfa versus timothy
based diets (p0.021). The serum calcium levels
of the timothy fed group were actually higher
than the serum calcium levels of the alfalfa fed
group (Table 4).
Introduction
Table 1 Guaranteed Analysis comparisons between
Purina rabbit cow, Labdiet HF (high fiber), and
Oxbow Bunnybasics T. Note the increased fiber
and decreased calcium in Oxbow. For this study,
LabDiet was chosen as the alfalfa diet because it
was already used at our institution. While there
are other timothy diets available (such as
Kaytee), Oxbow was chosen due to its popularity
and reputation with exotic animal veterinarians.
The 2nd edition of Laboratory Animal Medicine
suggests that rabbits prefer a diet that is low
in fiber and high in protein however, it is
widely recognized that rabbits require high fiber
diets to maintain proper GI health.
Historically, rabbit diets available both in the
pet trade and in the laboratory consisted
primarily of pellets. In the pet trade alfalfa
based pellets were offered with hay, vegetable,
and fruit supplementation. It is now widely
recognized by exotic animal veterinarians that
rabbits should be fed primarily a hay-based diet,
with limited pellet supplementation. In response
to this change, additional dietary options have
become available in recent years, including
timothy based pelleted diets that are higher in
fiber than alfalfa diets. In addition,
manufacturers are providing high fiber alflafa
based pelleted diets, which are the diets
popularly used in the laboratory. However, while
these high fiber alfalfa based diets do contain
similar amount of fiber, the diets differ
significantly in calcium levels. Alfalfa meal
contains roughly double the calcium of timothy
meal and this is reflected in the guaranteed
analysis of each diet (Table 1).
Alfalfa Timothy
Mean 13.2 14.4
SEM 0.3 0.2
Table 4 means and standard error of serum
calcium levels (mg/dl) for test groups at initial
(1st) data point.
Animals Twelve 6-month-old New Zealand White
Rabbits (Western Oregon Rabbit Company) were used
for the study. All animals were SPF for
Pasteurella. They were singly housed in Lab
Products stainless steel six rack cages and
offered water ad libitum. Cage pans were changed
twice weekly and racks were cleaned once weekly.
Temperature in the room was with 10-15 air
changes/hour and humidity of 30-70. All
procedures were approved by the Portland VA
Medical Center Institutional Animal Care and Use
Committee in accordance with federal policy.
Protocol Animals were each fed 150 grams/day
of either Labdiet HF pellets or Oxbow
Bunnybasics/T pellets for 6 months. Blood was
obtained monthly via jugular or marginal ear vein
and sent either to Antech or analyzed in-house.
A full CBC and serum chemistry was obtained for
each animal. Urine was collected monthly via
free catch or cystocentesis and submitted to
Antech for urinalysis and urine calcium levels.
To minimize the number of rabbits ordered
specifically for this study, the rabbits
receiving Labdiet (standard diet offered to
rabbits at our institution) were already involved
in other investigators studies (antibody
production).
Acknowledgements
Urine calcium levels Monthly urine calcium
levels for test animals ranged greatly from 8.5
to 66 mg/dl. Normal range (as reported from The
Clinical Chemistry of Laboratory Animals) is 22.3
/- 8.3 mg/kg/24 hours. At first data points,
final data points, and cumulative means, there
was no significant difference between urine
calcium levels of the timothy and alfalfa groups
(see Table 5).
The authors would like to thank Stephanie Murphy,
DVM, PhD for her assistance in statistical
analysis.
Calcium is an ion involved in bone metabolism and
neurologic function. Calcium requirements vary
for rabbits, depending on life stage. For
growth, gestation, and lactation the requirements
are 0.4, 0.45, and 0.75, respectively. One can
observe that dietary availability of calcium in
alfalfa diets far exceeds even lactational
requirements. Literature suggests that rabbits
often have higher total serum calcium levels than
other laboratory species and that urinary calcium
levels vary with serum calcium levels.
Therefore, rabbits fed diets high in calcium
should have higher urine calcium levels.
References
Alfalfa Timothy
Mean 23.4 9.1
SEM 4.2 1.1
Table 5 means and standard errors of cumulative
monthly urine calcium levels (mg/dl).

1. The Clinical Chemistry of Laboratory Animals 2nd
   ed. WF Loeb and FW Quimby. Edwards Bros, Ann
   Arbor, MI. 1999.
2. http//rabbit.purinamills.com/
3. http//www.zootoo.com/smallpets_pellets/oxbowbunny
   basicst2
4. www.labdiet.com
5. http//www.wisconsinhrs.org/Articles/Pellet20Type
   s.htm
6. Laboratory Animal Medicine, 2nd ed. Fox, JG,
   Anderson LC, Loew, FM, and FW Quimby. Elsevier.
   2002.

Renal gross necropsy and histopathology There
were no pathological lesions observed in either
treatment group.
Statistics Statistical differences were
calculated using Two Analysis of Variance and
students t-test. All statistical analyses were
performed using SigmaStat Statistical Software,
Version 3.0 (SPSS, Inc. Chicago, IL).
CBC and serum chemistry results Additional
bloodwork and urinalysis for each animal was
unremarkable. No compromise in renal function
was observed on bloodwork.