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A non-invasive method for assessing stress in the chinchilla (Chinchilla lanigera).
Marina F. Ponzio a,b,*, Steven L. Monfort b, Juan M. Busso a, Viviana G. Dabbene c, Rubén
D. Ruiz a and Marta Fiol de Cuneo a.
aInstituto
de Fisiología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Córdoba,
X5000ESU, Argentina.
bConservation & Research Center, Smithsonian’s National Zoological Park, Front Royal, VA 22630, USA.
cCEPROCOR, Colonia Santa María 5164, Santa María de Punilla, Córdoba, Argentina.
INTRODUCTION
The chinchilla, a strictly nocturnal rodent, is a member of the suborder Hystricomorpha, in which
two distinct species are recognized: Ch. lanigera and Ch. brevicaudata. These animals produce
the most valuable pelts in the world, and both were once abundant in the central Andes of South
America. Excessive hunting for fur and habitat fragmentation greatly reduced the number and
distribution of individuals at the beginning of the twentieth century. Wild populations were
harvested over a prolonged period of time at a high rate and the consequences for the wild
populations were soon evident; today, Chinchilla spp. are almost extinct in the wild and they are
listed on Appendix I of CITES. Although protected, the number of individuals are still declining
but the reasons are poorly understood. It is evident that without active management, research
and conservation, wild chinchilla populations will almost certainly become extinct in the near
future.
Although native chinchilla are extremely rare, a hybrid produced by cross-breeding the two
chinchilla taxa has been domesticated, bred and selected for superior fur production for more
than 80 years. Thus, the domestic chinchilla is different from both of the wild species. Today the
chinchilla represents a peculiar wildlife paradox: no other furbearer is so common in captivity yet
so rare in the wild .
Research conducted in common nondomestic or domestic animal models can be extremely
useful for developing an improved understanding of the biology of their endangered counterparts.
Similarly, knowledge obtained from studies of farmed chinchilla is likely to be directly applicable
to their wild counterparts, and may be important for enhancing captive breeding efforts designed
to provide a hedge against extinction.
On the other hand, it is well-known that the “stress” associated with sub-optimal
housing/husbandry conditions can compromise animal health and well-being, and adversely
impact reproductive function in many wild and domestic species. Breeders of the domestic
chinchilla have commonly observed fur-chewing and inter-sexual aggression. This behaviour is
presented randomly in the facility population. There is no set pattern to the disorder, it is sporadic
and unpredictable, and diverse explanations have been argued about the cause, treatment and
cure. However, none of this theories has been scientifically proved and all have a great lack of
evidence. In recent years however, this kind of behaviors have been attributed to “captivity
stress” in other species.
Physiological measures of the stress response have typically relied upon the evaluation of serum
or plasma glucocorticoids. However, attempts to obtain repeated blood samples from chinchilla by
either venipuncture or chronic indwelling catheterization were unsuccessful, in part, because of
small vein size and their stress-susceptible nature. Very small amounts of blood were obtained
by other authors through orbital or peripheral venipuncture or tail tip laceration. Fortunately,
noninvasive fecal and urinary corticosteroid monitoring can now be used to assess adrenal status
in nondomestic species. Noninvasive approaches in the chinchilla could permit long-term
endocrine monitoring while avoiding the potentially stress-evoking stimuli of restraint and
translocation, as well as risks associated with repeated venipuncture, including vascular damage,
infection, and anemia.
*
Corresponding author. Instituto de Fisiología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Santa
Rosa 1085, X5000ESU, Córdoba, Argentina. Tel/Fax: +54-351-4332019. e-mail: mponzio@mater.fcm.unc.edu.ar.
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Noninvasive corticoid monitoring could be
Organism responses to stress: physiological
particularly useful for investigating the
bases
relationship
between
various
husbandry/management strategies and
Animal life is constantly is defied by adverse or stressful
factors, both internal or external. The term stress has been
physiological stress in the chinchilla, with
used to indicate a reaction of general alert shot by new or
a particular interest in the elucidation if
unpredictable events that threaten homeostasis, causing a
whether or not stress is a major factor
defensive reaction of the organism towards the potentially
influencing the appearance of fur-chewing
dangerous stimulus. A general consensus exists that the
animals. An improved understanding of
answers to stress are physiological answers that do not
represent per se a threat for the organism, nevertheless, if
these relationships may help animal
they are intense and are maintained in time they can
managers to develop more effective
jeopardize the general and reproductive health. The
captive breeding programs for both
pathological alterations would derive then from the attempts
domestic and wild chinchillas.
of the organism to obtain physiological and neurobiological
adaptations to maintain homeostasis. The leading factors
The overall objective of this study was to
can be as variable as: touching states, injury, immobilization,
demonstrate the validity of noninvasive
heat, food deprivation, etc. One of the neuroendocrine most
corticosteroid monitoring for evaluating
well-known and consistent responses of the organism
adrenal responsiveness in the chinchilla
towards stressful situations is the activation of the
for further studies on stress.
hipotalamus-hypoofiso-adrenal axis (HHA). Thus, if the
stresful stimulus is adecuate, the liberation of the hormone
Technical validation was demonstrated by
ACTH is induced, which as well increases the synthesis and
1) determining the time-course of urinary
secretion of corticosteroid hormones of the adrenal gland. A
and fecal metabolites excretion after
variation in the secretion of these hormones exists, being
injection of radioactive markers, 2)
CORTISOL or CORTICOSTERONE the predominant
corticosteroid secreted according to the species. These
investigating the identity and relative
hormones reinforce the actions of the central nervous
proportion of those metabolites, and 3)
system on the circulatory apparatus and contribute to
demonstrating
specificity,
sensitivity,
elevate glucose levels in blood before an emergency
accuracy and precision of available
situation, preparing the individual for the defensive reaction.
commercial determination kits.
The increase of corticosteroids varies with the intensity of
the stimulation and the fluctuations in the circulating levels
Physiological validity was established by
can be used as an index of the stress state.
demonstrating
a
cause-and-effect
relationship between the activation of the adrenal gland through the administration of ACTH, and
the corresponding excretion of urinary corticosteroid metabolites.
MATERIALS, METHODS AND RESULTS
TECHNICAL VALIDATION
Time and principal route of excretion
To determine the time-course of corticosteroid metabolite excretion, and the proportion of
metabolites excreted in urine versus feces, two males were given injections of radioactive
markers in order to be able to detect those after the body metabolization and excretion.
A total of 45.5 ± 11.3% (n=2) radioactive-metabolites was recovered in urine and feces within 82
h of isotope administration; of this, 86.9 ± 0.07% of metabolized radiolabel was excreted into
urine whereas only 13.1 ± 0.1% in feces. After isotope administration, peak radioactive metabolite
excretion occurred ~5-10 h and ~30 h later in urine and feces, respectively (Figure 1).
Characteristics and relative proportion of metabolites
The quantity and relative distribution of corticosteroid metabolites in urine and fecal were
determined after reverse-phase high pressure liquid chromatography. This technique is used to
assist us in revealing the nature of the metabolites excreted into urine or feces.
Because the vast majority (>85%) of corticosteroids were excreted in urine, fecal steroid
metabolites were not evaluated by chromatography. Chromatographic separation of unprocessed
chinchilla urine revealed at least four corticosteroid metabolites, mostly conjugated (or water
soluble) cortisol metabolites.
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Hormonal determinations
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Animal B
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Horas post-inyección
Figura 1: Excreción de metabolitos de
corticosterona radioactiva en orina y heces
en dos machos adultos de chinchilla
doméstica (Animales A y B). La flecha
indica el momento de la inyección (i.m) del
radioisótopo.
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7000
Animal C
RIA Corticosterona
EIA Cortisol
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Animal D
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Figura 2: Concentración de corticosteroides
urinarios en dos machos de Chinchilla
lanigera doméstica (animales C y D), antes y
después de la inyección exógena de ACTH
en gel a tiempo 0 (flecha).
Inmunoreactividad metabolitos cortisol (ug/mg Cr)
Radioactividad en orina (CPM x 103)
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500
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Inmunoreactividad metabolitos corticosterona (ng/mgCr)
DISCUSSION
The route of excretion (proportion excreted in urine
vs. feces), the excretion lag-time (time from
appearance in blood circulation to excretion in
urine/feces), and
metabolic form
of excreted
corticoids differ between species. Therefore, the aim
of our study was to obtain basic knowledge about the
metabolism and excretion of
urinary and fecal
corticosteroids in the chinchilla. This information is an
essential prerequisite for developing a valid method
for noninvasively assessing adrenal activity (and
therefore stress) in this species.
Bolus injection of radiolabeled steroid, differential
extraction and subsequent chromatographic analysis
revealed that the majority (>85%) of corticosterone
metabolites were excreted in urine, after an excretion
lag-time of approximately 5-10 h. The vast majority of
immunoreactive corticosteroids were excreted as
conjugated forms of cortisol, and to a much lesser
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600
0
Inmunoreactividad metabolitos corticosterona (ng/mg Cr)
Comparison of the two immunoreactive profiles
revealed that the cortisol EIA (maximum peak, 600
ng/ml) detected more immunoreactivity than the
corticosterone RIA (maximum peak, 82 ng/ml). In
other words, the cortisol EIA detected 25-fold more
immunoreactivity (~800 ng/ml cortisol) compared to
the corticosterone RIA (~30 ng/ml corticosterone).
PHYSIOLOGICAL VALIDATION
ACTH injection
To determine the feasibility of detecting acute
increases in adrenal activity (as stress response) via
excreted corticosteroid metabolites, two adult males
were injected once with gel ACTH. Urine was
collected at approximately 4-h intervals, for 2 d
before and 4 d after ACTH administration. After
injection, urinary corticosteroid immunoreactivity
peaked (~4-fold above baseline) 5-10 h post-ACTH
administration. Profiles were similar in both males
(Figure 2).
Temporal excretion patterns in urinary cortisol were
similar, and peak cortisol immunoreactivity was
elevated ~7-fold higher than baseline concentrations.
Again, despite temporal similarities in excretion
patterns, peak cortisol immunoreactivity (animal C,
3985.9 µg/mg de Cr; animal D, el 5863.9 µg/mg de
Cr) was more than 3,000-fold greater than
corticosterone immunoreactivity (animal C, 57.4
ng/mg de Cr; Animal D, 250.7 ng/mg de Cr).
orina
heces
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3
Radioactividad en orina (CPM x 10 )
Animal A
700
Radioactividad en heces (CPM x 10 )
800
Inmunoreactividad metabolitos cortisol (ug/mg Cr)
Excreted corticosteroid metabolites determinations
were performed using commercial kits of
corticosterone RIA and cortisol EIA.
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extent, of corticosterone. Physiological validity was demonstrated by establishing a ‘cause-andeffect’ relationship between the administration of exogenous ACTH, and the subsequent excretion
of urinary corticosteroid metabolites. Overall, these results confirmed that urinary corticosteroid
metabolites provided a valid and feasible measure to noninvasively monitor changes in adrenal
activity in response to stress situations.
Cortisol (or its conjugates), were the predominant corticosteroid forms excreted after adrenal
activation in the chinchilla. Despite the finding that urinary cortisol metabolites were excreted in
much greater quantities than corticosterone metabolites, both measures were useful for tracking a
temporal increase in adrenal activity after the administration of exogenous ACTH. Nevertheless,
increased immunoreactivity detected using the cortisol EIA suggests that this immunoassay is
probably a more appropriate tool. It is clear, however, that whichever method (i.e., cortisol EIA or
corticosterone RIA) is employed, it is important to carefully characterize ‘baseline’ excretory
patterns to account for individual-animal variation.
The chinchilla has been severely overexploited by humans, and the native species are on the
brink of extinction. However, at present only a few studies have focused on its reproductive
physiology, and no studies have examined the interrelationships between animal well-being,
stress and reproductive fitness. The availability of the method validated in the present study will
improve our understanding of stress physiology in the chinchilla.
Acknowledgements
We are grateful for the technical assistance provided by staff of the Conservation & Research
Center’s Endocrine Research Laboratory. Animals were kindly provided by ACRICHI (Córdoba,
Argentina). Pelleted chinchilla food was provided by Daniel Melchert (Cargill, Cordoba).
Financial support was provided by the Agencia Córdoba Ciencia S.E, SeCyT-UNC, FONCyT 0505254, Friends of the National Zoo y the Scholarly Studies Program of the Smithsonian Institution,
Fundación Antorchas and the Chinchilla Industry Council.