I conducted research at the Sierra Nevada Aquatic Research Laboratory (SNARL) near Mammoth Lakes, CA. I live-trapped pregnant females from three sites near SNARL and housed them in a laboratory building where they gave birth and reared their young. Mothers were housed singly in stainless steel cages (61 × 45 × 35 cm) that included a 28 × 20 × 20 cm nest-box. I gave animals LabDiet mouse chow (no. 5015) and water ad libitum.
Starting around the age they would emerge in the field, I placed juveniles in a large, dark wooden box (0.61 m tall × 0.61 m deep × 0.91 m wide) and presented them with an acoustic stimulus in the middle of an otherwise silent 10-min period, with blood collected immediately after. I presented four categories of auditory stimuli: U. beldingi
whistles and trill alarm calls, U. beldingi
squeals (vocalizations made by juveniles during play) and a silent control (the latter two were used to record responses to common auditory stimuli that are not associated with predators). Digitized calls were played from a Sony TCM-5000 tape player connected to a Mineroff SME-AFS speaker suspended approximately 0.5 m high inside the box. All calls were presented at equal amplitudes. Additional details on the stimuli are in Mateo & Holmes (1997)
Littermates were tested in pairs, with one stimulus per day in a randomized order. Animals were lightly anaesthetized (10–20 s exposure) with isoflurane inhalant before drawing a blood sample (300–400 µl) from a saphenous vein within 3 min of removal from the box. Tests were conducted between the hours of 15.00 and 19.00 to minimize diurnal fluctuations in cortisol. Blood was refrigerated for an hour before being centrifuged for 10 min, and serum was stored at –15° C for four to six weeks, after which it was stored at –80°C until assayed.
Testing ended when I had one blood sample for each of the four stimuli or when juveniles reached 35 days of age. In some cases I was unable to collect enough blood in 3 min, or there was insufficient serum to assay. In 2006, I tested 26 juveniles from seven mothers (13 males, 13 females; obtaining at least one sample from eight males and seven females); I collected complete samples (trill, whistle, squeal and silent) for five males and three females. In 2008, I tested 18 juveniles from six different mothers (10 males, eight females), although no samples were obtained from one male; complete samples were collected from four males and five females.
I used 125
I-cortisol Corticote radioimmunoassay kits (MP Biomedicals, CA, USA) and assayed samples in duplicate. The sensitivity of the assay is 0.07 µg dl−1
. Two control samples, each made by pooling faecal extracts from five animals, were analysed in every assay (the ‘low’ pool, approx. 60–70% binding and the ‘high’ pool, approx. 20–30% binding; Mateo & Cavigelli 2005
). Across eight assays, mean intra-assay coefficients of variation for the assays were 11.72 per cent for the low pool and 5.98 per cent for the high pool. Mean inter-assay coefficients of variation were 6.48 per cent for the low pool and 6.72 per cent for the high pool.
Cortisol values were normally distributed. Using an individual's cortisol concentration following the silent stimulus as its baseline, I calculated the per cent change in its response to the whistle, trill and squeal, relative to the baseline, to standardize responses within individuals and to facilitate comparison across individuals. Because multiple individuals from a litter were used, I then averaged the cortisol responses by juveniles to each stimulus type for each litter. I next tested the null hypothesis that the per cent difference (also normally distributed) was not significantly different from zero using a one-sample t-test; one-tailed tests were used because responses to sounds were a priori expected to be higher than responses to silence. Finally, I analysed the data with a repeated-measures ANOVA using data from 17 individuals with serum samples for all four stimuli, after averaging the data for each litter (seven mothers contributing 1–4 juveniles each; average 2.42).