*3.1. Corticosterone Monitoring*

Mean baseline concentrations of excreted CM in the examined flocks were 43 ng/g and 66 ng/g for Variant I and Variant II, respectively. Overall, birds of Variant II had higher baseline values than birds of Variant I (Table 2). However, differences were not significant (effect: −18.6; 95% confidence interval (CI): ( −45.3; 9.1)).

**Table 2.** Comparison of baseline levels (mean ± SEM) of corticosterone metabolites measured in transport Variants I and II in H&N Super Nick (HNS) and H&N Brown Nick (HNB) animals, and in total for both layer lines.


HNB individuals had significantly lower baseline values than HNS birds (effect: −37.8; 95% CI: (−45.4; −30.3)). Three flocks showed significant deviations from mean baseline values in Variant II: two flocks with significant above-average values (D2 and D7) and one with significantly lower values (D6). Any effect that crossed zero did not significantly deviate from the mean baseline value in this flock (Figure 1).

**Figure 1.** Linear mixed-effects model. Effect of the flock on the baseline concentrations of corticosterone metabolites in either two transport variants, modeled with the flock as an unstructured random effect for the model constant, and with the transport variant and the layer line as fixed effects. The figure shows estimated values (circles) with their 95% confidence intervals (bars) of the effect of flock on baseline concentrations. Estimates that do not cross zero deviate significantly from the baseline concentration.

The transit from the rearing farm to the farm of the laying hens resulted in higher mean concentrations of CM compared with the mean baseline values. The highest values were found immediately on arrival (0 h). The mean concentration at 0 h was 173 ng/g and 323 ng/g for Variant I and Variant II, respectively. CM concentrations decreased rapidly during the 0–6 h interval after transportation. Variant I showed an increase during the 6–12 h interval, followed by a steady decline. Values for Variant II were slightly increased at 24 h, 48 h, and 72 h, and slightly decreased at 34 h and 58 h, with the additional sampling times considering the circadian rhythm (Table 3).


**Table 3.** Minimum (Min), maximum (Max), and mean concentrations ± SEM of corticosterone metabolites (CM) after transportation in two transport variants.

We found no significant difference in CM concentrations between Variant I and Variant II, because the overall trend was similar, and confidence intervals overlapped strongly (effect: −0.208; 95% CI: (−0.567; 0.163)) (Figure 2).

**Figure 2.** Linear mixed-effects model. Effect of time after transportation (hours) on concentrations of corticosterone metabolites (CM), considering the flock as a random effect, and the transport variant and the layer line as fixed effects. Estimated temporal progression of CM concentrations, shown as estimated effects (solid lines) and 95% confidence intervals (shaded areas) for each transport variant.

Significant differences in CM concentrations were found once again between the layer lines. HNS had higher values than HNB (effect: −0.286; 95% CI: (−0.334; −0.238)). Furthermore, considerable variations among flocks were found. Significant above-average values could be measured for one flock of Variant II (D7), significantly lower values for three flocks of Variant II (D5, D3 and D4), and one flock of Variant I (N4) during the 0–72 h interval. Any effect that crosses zero does not significantly deviate from the mean CM concentration in this flock (Figure 3).

**Figure 3.** Linear mixed-effects model. Effect of the flock on concentrations of corticosterone metabolites (CM) 0–72 h after transportation in either two transport variants, modeled with the flock as an unstructured random effect for the model constant, and with the transport variant and the layer line as fixed effects. The figure shows estimated values (circles) with their 95% confidence intervals (bars) of the effect of flock on CM concentrations. Estimates that do not cross zero deviate significantly from the mean value.

The ratio of CM concentrations after 72 h to CM baseline values showed that most CM concentrations did not return to baseline values in both transport variants (Figure 4). For both layer lines, we could not find a significant effect of transport variant on the return to baseline values (effect: 0.43; 95% CI: (−2.41; 3.26) and −1.49; 95% CI: (−4.34; 1.37) for HNS and HNB, respectively).

**Figure 4.** Ratio of concentrations of corticosterone metabolites (CM) after 72 h to CM baseline concentrations in two transport variants. Thick black lines show mean ratios, boxes represent upper and lower quartiles, whiskers represent 95% confidence intervals, and dots show outliers. A mean ratio above zero indicates an increase in CM concentration relative to the baseline value, whereas a mean ratio below zero indicates a decrease.

#### *3.2. Hen-Human Relationship: Touch Test*

With the touch test, we evaluated the hen–human relationship based on the approach and avoidance behavior of the birds between flocks. To evaluate whether this behavior was reflected in the measured CM concentrations, we compared CM concentrations between hens that stayed an arm length away from the examiner, and those that could be touched. An increase in CM concentration by one unit (1.0 ng/g) resulted in a significantly greater number of hens that could be touched (effect: 0.004; 95% CI: (0.001; 0.006)). In addition, a few differences in approach and avoidance behavior between flocks were found: One flock (D5) had significantly fewer hens that could be touched compared with three other flocks (D2, D6, and N2).
