A great deal of attention is paid to glucocorticoid levels in connection with the conservation of animal species in the wild or in captivity. However, monitoring the stress levels of laboratory test animals such as mice and rats can also be critical to understanding the results of the experiments in which they are used. To date, the majority of CORT assessment in laboratory animals is done with blood samples. CORT measurements in blood samples do provide a snapshot of stress levels at a moment in time, however, this snapshot can be influenced by random events unknown to the researchers, such as a squabble with cage-mates, and also by the stress of handling and of the blood draw itself. While blood draws remain a powerful tool for assessing acute stress response, they may not provide the most accurate reflection of the levels of chronic stress from housing situations or experimental protocols etc. that might be most of interest for researchers engaged in longer term studies.
Measuring glucocorticoids from non-invasive samples such as feces or urine removes sample collection as a potential source of stress and allows researchers insight into the chronic life stress an animal might be experiencing. Since this type of stress is more likely to influence the results of long term studies than acute situational stress response, there can be significant benefits to both the animals and the scientists. The fact that most laboratory rodents are housed in social cages does complicate the measurement of stress by feces or urine. In the past, researchers have gotten around this obstacle by reporting stress levels as an average for the animals housed in a single cage, but this is a less than ideal arrangement because an overly stressed animal that maybe should be removed from a study may be missed.
A recent paper by Cinque et al. used Arbor Assays Corticosterone EIA kit (K014-H) to validate a new fecal sample collection protocol in which individual rats were gently removed from their cages and handled briefly (less than 2 minutes) in towel until a fecal sample was obtained. For validation purposes, the test animals were exposed to one of two stressful stimuli – either a novel enriched environment, or predator urine. Samples were collected at the same time of day, for several days, and immediately frozen until the time of assay. As expected, both stimuli resulted in elevated levels of corticosterone over baseline. Corticosterone levels in the rats exposed to predator urine peaked relatively quickly representing the acute stress of a potential predator nearby, while corticosterone levels in the rats exposed to the mild chronic stress of the enrichment environment rose more slowly, not reaching significance until 72 hours after initial exposure. A separate experiment, comparing fecal samples collected twice a day (8 am and 8pm) confirmed a circadian rhythm for fecal corticosterone metabolites with levels being higher in the samples collected in the morning and lower in those collected in the evening. The authors were also able to show parallelism between pools of male or female fecal extracts and a dilution series of the corticosterone standard.
This study and others like it demonstrate that sample type, method of sample collection, and timing of sample collection all influence the levels of glucocorticoids measured. Relatively small difference in experimental design, such as the timing of sample collection can impact results. It is important to understand the implications variables and to design experiments such that the data generated are most relevant to the theory being tested.