Summary of the treatment procedures used in this study.

<p>The measurement of endogenous glucose production (EGP) was performed by administering [6-³H]glucose as a tracer and periodically drawing blood samples over 180 minutes. Animals in the “field sedated” study group were all measured at the rookery. Weanlings and yearlings in the “handling manipulation” group were each measured twice over two consecutive days. Weanlings were studied at the rookery while yearlings were transported to an animal holding facility for study.</p

Example of [6-³H]glucose clearance curves used to calculate endogenous glucose production (EGP).

<p>Curves are shown for one weanling and one yearling with and without the use of anesthetic agents using the same tracer dose. The lower specific activity observed in the weanling under physical restraint compared to chemical sedation indicates increased dilution of the label from higher rates of EGP under physical restraint. Equivalent ³H doses were administered to each seal, 100 µCi. dpm—disintegrations per minute.</p

Epinephrine concentration in handling manipulated seals.

<p><b>A</b>) Epinephrine concentrations were generally stable during procedures except in physically restrained weanlings. These restrained seals had elevated epinephrine concentrations at the beginning of the procedures; “<b>+</b>” and “<b>*</b>” indicate significant difference from initial epinephrine concentration in physically restrained weanlings and unrestrained yearlings, respectively (pairwise post-hoc t-test, p<0.05). Error bars are standard errors. <b>B</b>) The lowest epinephrine AUC values occurred while study animals were chemically sedated in both weanlings (paired t = 2.9, p = 0.045) and yearlings (paired t = 2.9, p = 0.03). Central horizontal lines indicate the median of each group; whiskers extend to data points within 1.5 times the interquartile range from each box.</p

Glucose response in handling manipulated seals.

<p><b>A</b>) The average glucose levels in physically restrained weanlings during the EGP measurement were significantly higher than the other groups (F_{3, 11} = 15.5, p<0.001). Error bars represent standard errors. <b>B</b>) Physical restraint significantly increased EGP (*) in weanlings (paired t = 3.1, p = 0.04) but there was no difference in EGP between chemically sedated and unrestrained yearlings (paired t-test, p>0.05). Central horizontal lines indicate the median of each group; whiskers extend to data points within 1.5 times the interquartile range from each box.</p

Relationship between EGP and cortisol levels.

<p>EGP did not vary with cortisol release among all study groups (F_{1, 14.7} = 1.0, p = 0.33). Note that the axes are not consistent between graphs, <b>A</b>) all study animals shown; <b>B</b>) adults, and <b>C</b>) juveniles, because the handling manipulated seals had a much larger range in cortisol AUC values than did field sedated seals. Dashed lines in <b>C</b>) indicate significant trends within the physically restrained and late weaning groups alone.</p

The anesthetic doses used during selected procedures.

<p>The total drug doses are reported as the mean and (sd). The induction dose was equal parts tiletamine and zolazepam—values shown are for each. For each agent the total dose, in mg or µg, and mass-specific doses are reported. The induction dose was administered in a single intramuscular injection; ketamine and diazepam were administered intravenously periodically over 3–3.5 hours of sedation. Data of anesthetic doses for adult males are not reported.</p

Cortisol concentrations during handling.

<p>The average cortisol concentration at each sample time within each study group; error bars represent standard errors. Note that the y-axis scales are different between the top and bottom graphs. RM ANOVA followed by pairwise post-hoc t-tests were used to test for significant differences from initial cortisol concentration. <b>A</b>) Handling manipulated group—physically restrained weanlings and chemically sedated yearlings showed increased cortisol levels during sampling. “+” and “*” indicate significant differences from initial (time = 0) cortisol value for physically restrained weanlings and chemically sedated yearlings, respectively (pairwise post-hoc t-test, p<0.05). <b>B</b>) Weaned pups early and late in post-weaning fast—late in the post-weaning fast, pups showed increased cortisol concentrations after 100 minutes of chemical sedation. “*” indicates significant difference from initial cortisol value (pairwise post-hoc t-test, p<0.05). <b>C</b>) Adult females early and late in lactation and after molting and <b>D</b>) adult males early and late in the breeding season and after molting. There was no significant difference in cortisol concentration with sample time among the adult samples.</p

Cortisol AUC value for each study group.

<p>The total cortisol present during the sampling period (cortisol AUC) for each study group; symbol and color coding matches that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038442#pone-0038442-g003" target="_blank">Figure 3</a>. Groups without overlapping letters were significantly different (p<0.05). See text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038442#pone-0038442-t001" target="_blank">Table 1</a> for additional descriptions of study groups. Central horizontal lines indicate median of each group; whiskers extend to data points within 1.5 times the interquartile range from each box.</p

Evoked potentials and analysis.

<p><b>A.</b> ABR measurement data of subject FR02. The evoked potentials at 16_sp-test; <b>B.</b> Results of the F_sp-test (bar plot, estimated variance of the ABR and the background noise as critical value for the test) and visual determination of the hearing threshold are indicated to the right of the panel. The waveform is classified as to whether the ABR is detected (o) or undetected (x) for F_sp and visual verification, respectively.</p

Spectra of the stimulus tone pips.

<p>Spectra of the generated (2-1-2) tone pip stimuli for the desired centre frequencies measured with the artificial ear. The peak frequencies showed a shift to frequencies lower than the desired peak frequencies for tone pips with centre frequencies ≥5.6 kHz. Signal distortion worsened and the bandwidth became more asymmetric relative to the centre frequency as the centre frequency of the tone pip increased.</p