Fecal corticosterone metabolite levels in two closely related rodent species in a sub-Mediterranean environment

Glucocorticoids regulate many physiological functions and play an important role in coping with challenging stimuli. The non-invasive assessment of glucocorticoids is increasingly used as a tool to evaluate individual and population health status in wild animals. Given the crucial role of rodents in forest ecosystems, it may be useful to study the glucocorticoid profile of these species to find possible links with the environmental characteristics in which they live and facilitate the development of biodiversity management plans. We studied two closely related species that are the main representatives of the ground-dwelling rodent communities in sub-Mediterranean forested areas: Apodemus sylvaticus and A. flavicollis. Fecal corticosterone metabolite (FCM) levels of animals captured in a Mediterranean agroforestry system were assessed. We found that A. sylvaticus males excreted lower FCM levels than females, while A. flavicollis males showed higher FCM levels than females. Males of the two species excreted similar FCM levels, while higher FCM levels were recorded in A. sylvaticus females than in their A. flavicollis counterparts. The FCM levels in both species were similar between breeding conditions, seasons, and habitat types. The results of our exploratory investigation suggest that traditional silviculture may not trigger the activity of the hypothalamus-pituitary-adrenal axis. Further studies are required for a more detailed examination of how environmental factors affect FCM levels. Long-term studies may disclose possible effects of interannual environmental factor variability in ground-dwelling rodents.

Methods
To evaluate the effects induced by different habitat types on mouse FCM levels we selected fifteen sampling sites within the three main habitat types: coppiced deciduous woodlands, conifer plantations and long-fallow fields.

Trapping sessions were conducted every other month from September 2011 to September 2012. In each sampling site, handmade LOT (Locasciulli Osvaldo Trap) live traps (9 x 8 x 23 cm) (Locasciulli et al. 2015), were placed on a 60 x 60 m grid. The grids were placed at least 100 m away from the habitat border. The minimum distance between two grids was around 500 m. The traps were equipped with nesting material (hemp) and baited with sunflower seeds, apples, and peanut butter. In each session, the traps were checked every morning for three consecutive days.

To evaluate the effects induced by trap confinement on mouse FCM levels and post-excretion FCM stability, opportunistic trapping events were carried out using food-baited L.O.T. traps placed in transects (20 traps, 10 m spacing). In particular, to test if the time mice spent in the traps was longer than their gut transit time, we added shredded colored waxes (Giotto be-bè®, Fila, Spain) to the bait as indigestible nontoxic markers (Serres-Corral et al. 2021). Traps remained active for three consecutive nights and were checked every morning.

The use of shredded colored waxes made it possible to distinguish the fecal boluses excreted in the trap before the digestion of the bait (i.e. boluses excreted in the 8-10 hours following capture) from those excreted afterwards. Almost all of the animals captured (around 90%) excreted both uncolored and colored fecal boluses inside the traps. The FCM levels of uncolored and colored fecal samples were assessed separately to compare their values (A. sylvaticus: 10 males and 10 females; A. flavicollis: 10 males and 9 females).

 A possible influence of factors such as temperature, humidity, and bacterial enzymes on the types and concentrations of fecal glucocorticoid metabolites after defecation has been described in some mammal species (Touma & Palme 2005). For this reason, certain studies assessing FCM levels in wild rodent species (e.g. Navarro-Castilla et al. 2014; Navarro-Castilla & Barja 2014) chose to collect only fresh fecal boluses (i.e. only feces with a soft texture) to avoid any issues regarding hormone metabolite stability. However, no data are available on the post-excretion stability of hormone metabolites in mice. To evaluate post-excretion FCM level stability, we collected fresh boluses excreted by the mice during handling for identification. The boluses of different animals from both species and sexes were mixed to obtain several sample pools (n = 6) for statistical analyses. Each pool was mixed to be homogeneous and five aliquots of 100 mg were taken from each pool for sampling. One aliquot from each set was immediately frozen at -20 °C while the others were left at ambient temperature for different time periods (6, 12, 18, 24 h) before being stored at −20 °C until hormone extraction was carried out.