**3. Results**

Average sIgA and cortisol concentrations for the three collection periods are summarized in Table 1. The highest average sIgA and cortisol concentrations were observed in samples collected at 06:00 h, while the lowest values occurred between 18:00 and 22:00 h, respectively. Individual concentrations of every time point is provided in Supplementary Materials Table S1.


**Table 1.** Daily and overall mean ± standard error of the mean (SEM) concentrations and ranges of salivary immunoglobulin A (sIgA) and cortisol in 10 Asian elephants (n = 5 male, 5 female).

a,b Means within rows with different superscripts are significantly different for each biomarker (*p* < 0.05).

Average sIgA and cortisol concentrations by sex are summarized in Table 2, with no di fferences observed at each time point (sIgA: *p* = 0.57, Cortisol: *p* = 0.73).


**Table 2.** Comparison of overall mean (± SEM) concentrations of salivary immunoglobulin A (sIgA) and cortisol between sexes (n = 5 males, 5 females) throughout three, 24 h periods.

From the GLS model, the e ffect of time was significant for sIgA (*p* = 0.0001), but only approached significance for cortisol (*p* = 0.06). There was an e ffect of collection day (sIgA; *p* < 0.0001, cortisol; *p* = 0.0235) for both biomarkers. Even though no e ffect of time was found for cortisol in the model, post hoc comparisons using the Tukey's honestly significant di fference (HSD) test indicated that mean sIgA concentration at 06:00 h was higher than that at 10:00 h (*p* = 0.002) and 18:00 h (*p* = 0.0001). By contrast, mean cortisol concentration at 06:00 h was only higher than that at 22:00 h (*p* = 0.0373). All data were used to construct a trend line using loess regression analysis. Mean and standard deviation (SD) are

presented in Figures 1 and 2 as well as the trend line for IgA and cortisol concentrations. Quartic (*p* = 0.04) trend effects for sIgA (Figure 1), and linear (*p* = 0.002) trend effects for cortisol (Figure 2) were evident throughout the 24 h cycle.

**Figure 1.** Scatter plot indicating quartic circadian pattern of sIgA concentrations in 10 Asian elephants. Blue dots represent the mean concentration of each time point. Red error bars represent the standard deviation. Blue line represents the trend line, and the shaded area is a 95% confidence interval of the mean.

**Figure 2.** Scatter plot indicating linear circadian pattern of cortisol concentrations in 10 Asian elephants. Blue dots represent the mean concentration of each time point. Red error bars represent the standard deviation. Blue line represents the trend line, and the shaded area is a 95% confidence interval of the mean.

sIgA and cortisol patterns of representative individuals depicting quartic and linear effects of the mean are shown in Figures 3 and 4, respectively. However, there was considerable variability and not all elephants followed clear patterns, as indicated in Figure 5. sIgA and cortisol patterns of all individuals is provided in Supplementary Materials Figures S1 and S2. There were missing samples at certain time points because of insufficient volume of saliva for analysis due to a combination of agitated elephants and human error in the collection process (Figures 4 and 5). There was only a weak, non-significant positive correlation (r = 0.099; *p* > 0.05) between sIgA and cortisol.

**Figure 3.** Representative individual profiles showing quartic circadian IgA trends in one male (**left**) and one female (**right**) elephant. Blue line represents the trend line, and the shaded area is a 95% confidence interval. Day 1 = red points, Day 2 = blue points, and Day 3 = yellow points.

**Figure 4.** Representative individual profiles showing linear circadian cortisol trends in one male (**left**) and one female (**right**) elephant. Blue line represents the trend line, and the shaded area is a 95% confidence interval. Day 1 = red points, Day 2 = blue points, and Day 3 = yellow points.

**Figure 5.** *Cont.*

**Figure 5.** Representative profiles of diurnal (**a**) IgA and (**b**) cortisol trends in one male (left) and one female (right) elephant that did not follow the typical or expected pattern. Blue line represents the trend line, and the shaded area is a 95% confidence interval of the mean. Day 1 = red points, Day 2 = blue points, and Day 3 = yellow points.
