A non-invasive method for assessing stress in the chinchilla



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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.
N
Organism responses to stress: physiological bases

Animal life is constantly is defied by adverse or stressful factors, both internal or external. The term stress has been used to indicate a reaction of general alert shot by new or unpredictable events that threaten homeostasis, causing a defensive reaction of the organism towards the potentially dangerous stimulus. A general consensus exists that the answers to stress are physiological answers that do not represent per se a threat for the organism, nevertheless, if they are intense and are maintained in time they can jeopardize the general and reproductive health. The pathological alterations would derive then from the attempts of the organism to obtain physiological and neurobiological adaptations to maintain homeostasis. The leading factors can be as variable as: touching states, injury, immobilization, heat, food deprivation, etc. One of the neuroendocrine most well-known and consistent responses of the organism towards stressful situations is the activation of the hipotalamus-hypoofiso-adrenal axis (HHA). Thus, if the stresful stimulus is adecuate, the liberation of the hormone ACTH is induced, which as well increases the synthesis and secretion of corticosteroid hormones of the adrenal gland. A variation in the secretion of these hormones exists, being CORTISOL or CORTICOSTERONE the predominant corticosteroid secreted according to the species. These hormones reinforce the actions of the central nervous system on the circulatory apparatus and contribute to elevate glucose levels in blood before an emergency situation, preparing the individual for the defensive reaction. The increase of corticosteroids varies with the intensity of the stimulation and the fluctuations in the circulating levels can be used as an index of the stress state.


oninvasive corticoid monitoring could be particularly useful for investigating the relationship between various husbandry/management strategies and physiological stress in the chinchilla, with a particular interest in the elucidation if whether or not stress is a major factor influencing the appearance of fur-chewing animals. An improved understanding of these relationships may help animal managers to develop more effective captive breeding programs for both domestic and wild chinchillas.

The overall objective of this study was to demonstrate the validity of noninvasive corticosteroid monitoring for evaluating adrenal responsiveness in the chinchilla for further studies on stress.



Technical validation was demonstrated by 1) determining the time-course of urinary and fecal metabolites excretion after injection of radioactive markers, 2) investigating the identity and relative proportion of those metabolites, and 3) demonstrating specificity, sensitivity, accuracy and precision of available commercial determination kits.

Physiological validity was established by 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.

Hormonal determinations

Excreted corticosteroid metabolites determinations were performed using commercial kits of corticosterone RIA and cortisol EIA.

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


T
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.
o 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).


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.

B
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).

olus 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 extent, of corticosterone. Physiological validity was demonstrated by establishing a ‘cause-and-effect’ 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 05-05254, Friends of the National Zoo y the Scholarly Studies Program of the Smithsonian Institution, Fundación Antorchas and the Chinchilla Industry Council.
Каталог: engl
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