*4.1. Corticosterone Monitoring*

Baseline values of CM concentrations and values measured between 0 h and 72 h after transportation did not show significant differences between the two transport variants. Instead, we found significant differences between the two layer lines in both baseline values and values measured after transportation. However, other studies found similar baseline plasma corticosterone concentrations in brown and white layer lines [33,40]. One study analyzed translocation stress in ISA Brown (name of hybride) hens for 36 h after a 1 h long transportation, and found the highest plasma corticosterone concentrations 4 h after transportation [23]. In contrast to this finding, the HNS and HNB hens of our study showed a rapid decrease in CM concentration during the first 6 h after transportation, but just a few returned to baseline values at the end of the study, which might be due to the novel environment. HNS had higher CM levels than HNB at all times in almost every flock. Fraisse and Cockrem [33] reported similar results after 15 min of repeated handling. White hens of their study also showed higher corticosterone levels than brown hens, but only for plasma corticosterone, whereas fecal CM concentrations did not differ between layer lines. At 9 h and 12 h after transportation, CM concentrations in Variant I of our study showed an increase from 61 ng/g at 6 h after transportation, to 96 ng/g and 127 ng/g, respectively, with an intermittent decrease at 10 h (69 ng/g), followed by a steady decline (Table 3). Samples at 9 h and 12 h were taken solely from Flocks N1 and N2; for Flocks N3 to N8, we reduced the sample collection to once at 10 h after transportation. During the investigation period of 72 h, CM concentrations never fell below the value measured 6 h after transportation. In Variant II, we found slight fluctuations of CM concentrations at 34 h and 58 h (additionally taken samples), indicating natural variation due to the circadian rhythm during a 24 h interval. De Jong et al. [41] found a plasma corticosterone peak at 4 h of the 8 h light period during a 24 h investigation on 5-week-old broilers that were fed ad libitum and showed low plasma corticosterone levels during the dark period for 12 h. This finding is contrary to the results from Variant II of our study because samples at 24 h, 48 h and 72 h were taken during the dark period (between 11:00 p.m. and midnight) and samples at 34 h and 58 h were taken during the light period (9–10:00 a.m.). Differences between individual flocks might be attributed to the so-called "passage effect": Management and processes of transportation, for example, differed. Further investigations are necessary to better understand these differences.

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

The relationship between animals and humans is an important aspect of animal welfare. Additional contact to humans can positively influence the hen–human relationship [28]. Studies on laying hens showed that additional positive contact with a person resulted in reduced fear toward this person [25,28,42] and in a decrease of plasma corticosterone levels [25]. The pullets of our study behaved contrarily to these findings: Pullets with increased CM concentrations were more likely to allow touch by the test person than pullets with low CM concentrations. Four flocks deviated from the average test results. However, these flocks did not show deviations in any of the other study parts. We therefore cannot relate the tameness of these flocks to other test results of this study.
