Non-invasive endocrine monitoring in the aardwolf
(Proteles cristatus) using faecal samples
Drs. C.M. Muilwijk
Faculty of Veterinary Science, Utrecht University, the Netherlands 2011
Four aardwolves (two males and two females) in South Africa were studied to indentify a suitable test system for
assessing gonadal and adrenocortical endocrine function based on faecal analysis during four months. It was
possible to determine the following classes of hormones in the faeces of aardwolves: androgen, progestagen,
estrogen and glucocorticoid metabolites. For androgen and progestagen metabolites a significant difference
between the females and males was measured. For a physiological validation of faecal glucocorticoid
metabolites, two aardwolves were darted and injected with exogenous ACTH. The GCM levels in the feaces
increased significantly within respectively 3-20 hours after the injection. This ACTH challenge showed that the
measured feacal glucocorticoid metabolites were relevant indicators of adrenal activity in aardwolves. It can
therefore be useful as a non-invasive tool for measuring a physiological stress response non-invasively in this
species.
INTRODUCTION
Although the current IUCN status
of the aardwolf (Proteles cristatus) is
“Least Concerned” [IUCN 2008], there is a
general lack of biological knowledge of
these myrmecophagous mammals. More
information of the reproductive
endocrinology and their behavior ecology
can be helpful to protect this species and
the vitality of their ecosystems.
For a successful investigation of
reproductive-endocrine relationships in the
aardwolf, collection of repeated samples
for hormonal evaluations is necessary.
[Schwarzenberger et al., 1996]. However,
collecting blood from free-living
aardwolves requires anesthesia, which is
usually stressful and dangerous for the
animal and also expensive and impractical.
Furthermore, physiological stress by
darting animals may obscure the normal
hormonal milieu. An alternative option is
using non-invasive sampling methods;
urinary, salivary, sweat, feathers, milk,
nails, hair and faecal sampling [Cook et al,
2000], which reduce pain and suffering for
the animals. Additionally, non-invasive
materials can be collected even daily for
longitudinal studies [Monfortet al., 2002;
Dloniak et al., 2003]. An extra benefit of
collecting faeces instead of blood is that a
pooled fraction of plasma hormones is
measured and not the fluctuations of
hormones in the blood. [Creel et al., 1996;
Goymann et al., 1999]. However, noninvasive measurements need to be
validated to ensure that the measured
compounds reflect circulating levels of the
biologically active target agents [Cekan,
1975; Schwarzenberger et al, 1996].
Alongside measuring the
reproductive hormones, it is also
interesting to learn more about the
physiological responses to environmental
and social stressors. Adrenal
glucocorticoids play an important role in
the homeostasis of an individual. If the
homeostasis of an animal is disturbed by
physiological or behavioral stressors, the
animal responses with the secretion of
glucocorticoids by the hypothalamicpituitary-adrenal axis (HPA axis)
[Ganswindt et al., 2010]. Because it is
impossible to predictably find wild animals
with elevated stress levels, the HPA axis is
triggered with a conducted
adrenocorticotrophic hormone (ACTH). To
measure whether the glucocorticoid levels
increase in the aardwolf after the ACTH
injection, faecal and blood samples were
collected both before and after the
injection.
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
In recent years, reliable noninvasive methods have been established for
assessing hormone metabolite levels in
faeces for a large number of mammalian
species [Schwarzenberger et al., 1996;
Palme-Möstl, 1997; Ganswindt et al.,
2003]. One of the species for which this
has been examined is the spotted hyena
(Crocuta crocuta), a close relative of the
aardwolf [Goymann et al., 1999; Dloniak
et al., 2004]. Because of the lack of
knowledge concerning the hormone
metabolite levels of aardwolves and the
proven usefulness of using faeces for
assessing the hormone metabolite levels in
relative species this method was chosen to
be applied to aardwolves. Although there
were some concerns whether this method
would work with this species. The aim of
this study was to investigate the hormone
metabolite levels of aardwolves by making
use of their faeces. The endocrine
parameters used in this research contain
the following hormone metabolites:
androgen, estrogen, progestagen and
glucocorticoid. Androgen (testosterone),
estrogen and progestagen (gestagen)
metabolites were compared between
females and males for validation.
Glucocorticoid metabolites were measured
in the faeces and blood for the ACTHchallenge. These hormones will give more
information about the various aspects of
the endocrine activity of this species.
months (8 to 40 °C) [Stenkewitz and
Kamler, 2008].
Study animals
For this study four radio-collared
aardwolves (Proteles cristatus), two males
(BWM08002, BWM09008) and two
females (BWF08006, BWF09009), were
used. Together with the spotted, brown,
and striped hyena, the aardwolf belongs to
the Hyaenidae, the second smallest family
of the Carnivora. Aardwolves have an
average body mass of 8 – 12 kilogram.
They are social monogamous but sexually
polygamous, primarily nocturnal, and
obligate termitivores feeding primarily on
harvester termites [Williams et al., 1997;
Taylor-Skinner, 2000].
Figure 1. BWM09008, one of the studied
Aardwolves.
The aardwolf is limited to Africa
and occurs in two discrete populations. The
southern population ranges over most of
southern Africa, extending just into
southern Angola, southern Zambia, and
southwestern Mozambique [KoehlerRichardson, 1990]. The northern
population extends from central Tanzania
to northeastern Uganda and Somalia, then
narrowly along the coast of Ethiopia and
Sudan to extreme southeastern Egypt
[Koehler-Richardson, 1990]. Our studied
animals belong to the southern population.
MATERIALS
Study site
This study was conducted between
November 2009 and March 2010 on
Benfontein Game Farm (BGF; 28°50′ S,
24°50′ E), near Kimberley in central South
Africa. Benfontein is 11.000 hectare and is
located in an area where Nama Karoo,
grassland and Kalahari Savanna meet. The
mean annual rainfall is 430 ± 127 mm.
There is a distinct cold and dry period
during winter months (–8 to 25 °C), and a
hot and rainy period during summer
METHODS
Faecal samples
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C.M. Muilwijk, Utrecht University
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
During the day all the radio
collared (Sirtrack®, Havelock North,
approx. weight 60g/collar) aardwolves
were tracked in their dens using VHFtransmitters. Almost every night at least
one aardwolf was followed by car. The
studied animals were not shy of the motor
vehicle and appeared undisturbed when
followed within 15 m.
In order to monitor faecal hormonal
metabolites, the aardwolf was followed
until it defecated. Bi-weekly faecal
samples were collected (n = 66). Before
these animals defecated, they dug a narrow
trench with alternating strokes of the
forepaws. Then the aardwolf turned around
and squatted over the trench. The first
defecation of the evening may be up to 8%
of the body mass of the animal [KoehlerRichardson, 1990]. The faecal samples
collected in this study were for the greater
part the first defecations of the evening.
The subsequent two or three defecations
are much smaller [Koehler-Richardson,
1990]. After defecating, the aardwolf filled
the hole with sand and usually deposited a
few scent marks before leaving. After the
animal had defecated and moved away, the
faecal sample was excavated using
disposable rubber gloves. A sample was
taken from the middle of the bolus to avoid
cross-contamination with urine or
contamination with other faecal samples in
the area. Subsequently, the sample was
transferred into a glass vial, immediately
placed on ice and was frozen at -20ºC until
extracted.
To measure whether the
glucocorticoid levels in the faeces increase
after the ACTH injection, faecal samples
were collected 48 hours before and 48
hours after the injection. The faeces
collected in the pre-injection period
provided a measure of baseline
glucocorticoid levels.
Two aardwolves, BWF08006
(female) and BWM09008 (male), were
darted using a CO2 powered dart gun
(Telinject, Germany) with 36 milligram
ketamine hydrochloride (Anaket-V®) per
kilogram bodyweight and 0,6 milligram
medetomidine hydrochloride (Domitor®).
Approximately 60 minutes after darting the
medetomidine was reversed with 3.0 mg
atipamezole (Antisedan®).
Both aardwolves were also injected
with procaine penicillin and benzathine
penicillin (Duplocillin®) at 15 IU per
kilogram bodyweight to avoid infections
from the dart wounds.
Figure 3. One of the studied aardwolves
(BWF08006) was injected intramuscularly with 10
IU of synthetic ACTH.
First blood was taken from the
anesthetized aardwolves. The next
injection was 10 IU of synthetic
adrenocorticotrophic hormone
(Synacthen® depot, Novartis, Australia)
intramuscularly. After 60 minutes another
blood sample was taken. Blood samples
Figure 2. Frozen glass vials with faecal samples.
ACTH challenge
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C.M. Muilwijk, Utrecht University
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
were taken to compare the immunoreactive
cortisol in blood with the glucocorticoid
metabolites concentrations measured in the
faecal samples collected both before and
after the ACTH injection for validation.
For the validation of the
reproductive hormones bi-weekly faecal
samples were collected of each aardwolf,
in order to improve the knowledge of the
hormone metabolite levels in aardwolves.
Two females and two males were studied
to compare the reproductive hormone
metabolites between the different sexes.
Faecal progestagen metabolites
were analyzed by EIA of 5α-pregnan-3βol-20-on as described by Heistermann et al.
[1997] who used the assay for determining
luteal activity in the African elephant
(Loxodonta Africana). The 5α-pregnan-3βol-20-on concentrations was quantified by
an EIA following Prakash et al. [1987]
using a polyclonal antibody against 5αpregnan-3β-ol-20-one-3-HS-BSA and 5αpregnan-3β-ol-20-one-3-HS-peroxidase
label. Sample concentrations are expressed
as µg/g organic dry content.
The EIA used in this study to
analyze GCM metabolite levels was an 11oxoaetiocholanolone first described by
Möstl et al. [2002]. This EIA detects GCM
with a 3α-hydroxy-11-oxo structure and
has successfully been validated for
monitoring adrenocortical activity in
various primate species, the African
elephant, and other mammals [Möstl et al.,
2002; Ganswindt et al., 2003; Heistermann
et al., 2006]. Sample concentrations are
expressed as ng/g organic dry content.
The enzyme immunoassays for
faecal androgen, estrogen, progestagen and
GCM metabolites levels were performed
on microtiter plates with a double antibody
technique according to the methods
described by Palme and Möstl [1997] and
Ganswindt et al. [2002].
For the measurement of GCM
metabolite levels in the blood samples a
commercially available Coat-A-Count®
Cortisol radioimmunoassay from Siemens
Medical Solutions Diagnostics was used.
This is a solid-phase RIA wherein 125Ilabeled cortisol competes for 45 minutes
with the cortisol in the patient sample for
antibody sites [Siemens Healthcare
Diagnostics, 2006].
Extraction and assay
Faecal samples were lyophilized,
pulverized and sifted using a mesh strainer
to remove fibrous material [Fiess et al.,
1999]. 0,2 g of the faecal powder was then
extracted with 3 ml 80% ethanol in water.
After vortexing for 15 minutes the mixture
was centrifuged for 10 minutes at 1,500 g.
The supernatant was transferred to a
micro-centrifuge tube for hormone
analysis. However, due to a highly variable
amount of mineral content in the collected
samples (49,5-92,5% of total dry weight),
the organic content of the samples was
determined by burning extracted samples
in a muffle furnace for 90 minutes at 450
ºC and expressed the measured hormone
levels as mass hormone/mass organic
content in extracted faeces.
Feacal androgen metabolites were
analyzed with a biotin-streptavidin enzyme
immunoassay (EIA) first described by
Palme and Möstl [1994]. Antibodies were
raised against 5α-androstane-3α-ol-17-oneHS. 5α-androstane-3,17-dione-thioether
was used as label and epiandrosterone was
used as standard. Sample concentrations
are expressed as µg/g organic dry content.
For the measurement of faecal
estrogen metabolites a biotin-streptavidin
EIA was used with antibodies against 17βoestradiol-17-HS and oestrone was used as
standard [Palme-Möstl, 1994]. Sample
concentrations are expressed as ng/g
organic dry content.
Data Analysis
An independent sample t-test was
used to test the androgen metabolite level
found in the faecal samples of the
aardwolves. The males and the females
were compared on significant differences
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C.M. Muilwijk, Utrecht University
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
in the means of their scores on the
concentration androgen metabolite levels
measured from the faecal samples.
For estrogen metabolites an
independent sample t-test was used as well
to compare the faecal concentration for
significant differences between the males
and females.
An independent sample t-test was
used to control the manipulation of the
progestagen metabolite level found in the
faecal samples. The male aardwolves and
the female aardwolves were compared on
their scores on concentration progestagen
metabolites measured from the faecal
samples.
For the measurement of the
glucocorticoid metabolite levels the
ACTH-challenge was accomplished. A ttest was used for the male (BWM09008)
and the female (BWF08006) in question to
compare the GCM metabolites levels in the
faeces before and after the ACTH
injection.
To measure the differences in
immunoreactive cortisol in the blood for
the specific male and the female, a paired
sample t-test was done to check for
significant differences pre and post
injection for each aardwolf who had an
injection. We crosschecked the
immunoreactive cortisol blood levels with
the GCM faeces metabolite levels for the
ACTH injection group to determine if
these were the same or not and in which
degree an increase was seen.
RESULTS
There were considerable
differences between the studied
aardwolves in terms of progestagen
(gestagen), androgen (testosterone) but not
in estrogen metabolites (Table 1).
Table 1 Average hormone levels of the studied animals.
Animal ID
Conc.
Conc.
Conc.
Conc. GCM
Progestagen Androgen
Estrogen (ng/g (ng/g org)
(µg/g org)
(µg/g org)
org)
BWF08006
BWF09009
BWM08002
BWM09008
34,364835
43,264464
6,502130
11,526497
9,012874
10,162596
20,517373
19,959667
54,110678
64,302506
73,443951
66,031999
960,111460
187,828617
193,363149
455,461890
for androgen metabolites for the male
samples (M=20,24) differed significantly
from the average for female samples
(M=9,50), (t(64)=-3,41, p=0.001).
Progestagen (gestagen)
Females had significantly higher
faecal progestagen metabolite levels
compared to males (Fig. 4). The average
progestagen metabolite level (M= 9.01) for
the male samples were significantly
different from the average progestagen
metabolite level for the female samples
(M=38,11) (t(64) = 4.90, p=0.000).
Estrogen
There were relatively marginal
differences between the studied animals in
faecal estrogen metabolites (Fig. 6). The
average estrogen metabolite level (M=
69,74) for the male samples were not
significantly different from the average
estrogen metabolite level for the female
samples (M=58,40), (t(51) = -1,72,
p=0.092).
Androgen (testosterone)
Males had significantly higher
faecal androgen metabolites levels
compared to females (Fig. 5). The average
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C.M. Muilwijk, Utrecht University
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
metabolite levels returned to baseline
within 44 hours (Fig. 7).
The injection yielded a significant increase
in faecal GCM metabolite levels for both
the male (t (13)= 2,68, p=.0.020) and for
the female (t (21)= 2,71, p=0.048).
For the two studied animals the
immunoreactive cortisol blood values and
average GCM faeces values pre- and post
ACTH injection were established (Table
2). Faecal samples values increased 5,9x
on average, for the blood values this was
5,5x on average.
By using a paired sample t-test we
determined if the blood values before and
after the ACTH injection were
significantly increased. The mean
beforehand (M=105,50) differed
significantly from the mean afterwards
(M=571,66) t (1)= -46,8, p=0.14.
ACTH challenge
The mean GCM metabolite levels
for the ACTH injection group (M=763,86)
differed significantly from the mean GCM
metabolite levels from the non-ACTH
injection group (M=190,41), t(64)=-2,23,
p=0.172. The glucocorticoid metabolites
level increased significant in the faecal
samples after the ACTH injection until 48
hours post injection for both the male
(BWM 09008) and the female
(BWF08006) (Fig 7). The ACTH injection
yielded peak GCM metabolite levels in the
faeces of 621% (female: 2129,92 ng/g org)
and 542% (male: 1242,53 ng/g org)
compared to pre-injection baseline. Mean
pre-injection levels were 343,04 ng/g
org and 229,28 ng/g org respectively for
female and male. Peak GCM metabolite
level for the female was measured in a
fresh faecal sample 3 hours and 12 minutes
after injection whereas peak glucocorticoid
metabolite level for the male was measured
20 hours and 18 minutes after injection.
For both the female and the male, GCM
Table 2. GCM blood and faecal values before and after ACTH injection.
Sample
Pre-injection
Post-injection
BWF08006
Blood
155,3 nmol/l
631,4 nmol/l
BWM09008
Blood
55,7 nmol/l
511,9 nmol/l
BWF08006
Faeces
343,04 ng/g org
2129,92 ng/g org
BWM09008
Faeces
229,28 ng/g org
1242,53 ng/g org
than in the males, but no significant
difference was measured. A more
comprehensive follow-up study should be
done to investigate this.
The elevated GCM metabolite
levels as a response to the ACTH challenge
suggests that the measured metabolites are
relevant indicators of adrenal activity.
However, the intensity of the
physiologically induced stress response
that we measured is different between the
two aardwolves. A likely explanation for
the mean peak of the female is that the
animal was stressed prior the injection.
DISCUSSION
The results showed that it was
possible to determine androgen,
progestagen and glucocorticoid metabolites
in the faeces of aardwolves.
As expected androgen metabolites
were significantly higher in the males than
in the females and progestagen metabolites
were significant higher in the female than
in the males. We also expected that
estrogen metabolites would be higher in
the females
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C.M. Muilwijk, Utrecht University
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
In spotted hyenas (Crocuta
crocuta), feacal glucocorticoid metabolites
peaked 26 ± 5 hours after ACTH injection
[Goymann et al. 1999]. This is similar to
our results on aardwolves, where feacal
GCM metabolites peaked approximately
20 hours after ACTH injection in the male
and between 3 - 24 hours in the female.
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CONCLUSION
In this study we demonstrated
progestagen, androgen, estrogen and
glucocoricoid metabolites levels in the
faeces of four studied animal and therefore
feaces collection can be used for noninvasive endocrine measurement in
aardwolves.
Progestagen metabolites were
significantly higher in the studied females
and androgen metabolites were
significantly higher in the studied males.
The elevated GCM metabolite
levels in the feaces as a response to the
ACTH challenge presented that the
measured metabolites are relevant
indicators of adrenal activity. It can
therefore be useful as a non-invasive tool
for measuring a physiological stress
response in this species.
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ACKNOWLEDGEMENTS
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Hodges JK. 2002. Assessment of testicular
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I express my gratitude to F.
Dalerum and A. Ganswindt from the
Mammalian Research Institute, University
Pretoria for their guidance, encouragement
and helpful advice during this project. I
would also like to thank M. Engelkes from
University Utrecht for her support during
the data collection. I thank M. Paris from
University Utrecht for giving me this
opportunity. Finally I would like to thank
De Beers Consolidated Mines for
permission to work on their property
Benfontein Game Farm.
Ganswindt A, Münscher S, Henley M,
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C.M. Muilwijk, Utrecht University
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
LEGENDS
Figure 4. Gestagen concentrations in the feaces of the studied aardwolves.
Figure 5. Androgen concentrations in the feaces of the studied aardwolves.
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C.M. Muilwijk, Utrecht University
Non-invasive endocrine monitoring in the aardwolf (Proteles cristatus) using faecal samples
Figure 6. Estrogens concentrations in the feaces of the studied aardwolves.
Figure 7. Responses of immunoreactive GCM concentrations in faeces following IM
injections of synthetic ACTH in two aardwolves.
7000
Conc. GCM (ng/g org)
6000
5000
4000
BWM09008
3000
BWF08006
2000
1000
0
-56
-44
-32
-20
-8
4
16
28
40
Hours since AcTH injection
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C.M. Muilwijk, Utrecht University