Comparison of different drying and storage methods on quantifiable concentrations of fecal steroids in the cheetah

Abstract

Fecal steroid analysis is a powerful tool that can provide important information on the health, physiology, and reproductive status of nondomestic species. However, studying free-ranging animals requires that feces be stored and transported from the collection site to the laboratory in a manner that prevents degradation or alteration of steroid metabolites. To determine the effects of different handling and storage methods on fecal steroids, 30 fresh fecal samples from five captive cheetahs were collected, thoroughly mixed, separated into aliquots, and processed (stored or dried) under different conditions. Concentrations of gonadal and adrenal steroid hormones were analyzed in feces stored frozen at -20°C or at room temperature in 95% ethanol. Both frozen and ethanol-stored aliquots were desiccated using a lyophilizer, solar oven, or conventional oven. The steroid values from aliquots stored and desiccated using the different methods were compared to those obtained using the optimal storage method of freezing at -20°C and desiccating in a lyophilizer (control). Concentrations of corticoid, estrogen, progestagen, and androgen metabolites in fecal extracts were quantified by radioimmunoassay. Androgen metabolite concentrations were not significantly affected (P > 0.05) by storage or drying methods. Fecal samples stored at room temperature in ethanol and lyophilized also had steroid concentrations that did not differ (P > 0.05) from controls. However, the concentrations of corticoid and estrogen metabolites were significantly lower (P < 0.05), and progestagen metabolites were significantly higher 216Terio et al.(P < 0.05) in samples desiccated in solar and conventional ovens without regard to storage method. These results suggest that storage of fecal samples at room temperature in ethanol is the best alternative to freezing for subsequent analysis of steroid hormone concentrations. Differences in measured concentrations of hormones in oven-desiccated samples could be due to hormone degradation or shifts in the immunodominant metabolite. Therefore, validation of storage and processing techniques should be included in the development of any new fecal steroid analysis methodology. Zoo Biol 21: 215-222, 2002.

Effects of fecal age and seasonality on steroid hormone concentration as a reproductive parameter in field studies

“…The origin of all these differences is the confluence of multiple causes pointed out by other authors: specificity of microbes in the feces that metabolize the fecal steroids, effect of temperature on steroid metabolite degradation, environmental moisture which allows the growth of microbes and fungi, change in metabolite affinity for antibodies, and the different chemical stability of each hormone (Matkovics 1972;Woods 1975;Neumann et al 2002;Terio et al 2002;Washburn and Millspaugh 2002;Millspaugh and Washburn 2004). Exhaustive condition-controlled laboratory metabolism studies, followed by precise identification of new metabolites, HPLC mass spectrometry, nuclear magnetic resonance, or infrared spectroscopy, are recommended to find out the precise hormone metabolism and degradation processes and the factors affecting them.…”

“…Because of microbial metabolism and other biochemical processes that occur over time, changes in humidity and temperature are probably the most influential environmental factors affecting steroid metabolite concentration in feces of free-ranging animals (Washburn and Millspaugh 2002;Terio et al 2002). Moreover, under field conditions, the fecal material undergoes changes in water content derived from its exposure to changing seasonal environmental conditions.…”

Concentrations of four fecal steroids in wild baboons: short-term storage conditions and consequences for data interpretation

“…Experiments on the effects of medium-term storage on fecal steroids (e.g., Khan et al, 2002;Terio et al, 2002;Washburn and Millspaugh, 2002) have lagged behind those for urinary steroids (e.g., Brown et al, 1995;Grant and Beastall, 1983;Kesner et al, 1995), despite the recognition that, due to the bacterial load in fecal matter, steroids are inherently less stable in feces than in urine , and in fact, steroid levels can change within hours following defecation if samples are not immediately preserved (M€ o ostl et al, 1999). This study considers the effects of short-term, weeks-long, storage conditions on quantifiable fecal testosterone (fT), glucocorticoid (fGC), estrogen (fE), and progestagen (fP) concentrations in wild baboons (Papio cynocephalus).…”

Effects of storage temperature and time on fecal progesterone concentration<br>in camel (Camelus dromedarius)

“…Thus, the increase of the steroid concentration could be the result of the antibody cross-reactivity with other hormonal metabolites created by microbial transformation during storage (Terio et al, 2002). However, samples that were dried and stored in room temperature did not have significantly different fecal progestagen from initial concentration at 7 and 15 days.…”

“…Other studies showed that ethanol has been used as a preservative for shortterm ambient temperature storage of fecal samples (Khan et al, 2002;Lynch et al, 2003). Furthermore, Terio et al (2002) suggested that storage of fecal samples at room temperature in ethanol was the best alternative to freezing for subsequent analysis of steroid hormone concentrations because it stabilized the concentrations of metabolites for progestogen and estrogen in cheetah for a period of up to 2 weeks. Other ambient temperature field storage techniques included drying samples.…”