*2.2. Corticosterone Monitoring*

To examine the effects of transportation and translocation on stress in each flock, corticosterone levels were measured non-invasively by extracting metabolites from bird droppings. For each sampling time point, 25 pullets of each genetic line were randomly caught from different tiers of the dimmed barn. To enable the collection of individual, spontaneously voided droppings, the pullets were placed separately in cleaned and disinfected plastic crates, and marked on their legs with a pen (Edding-Egg-Color-Pen, Wunstorf, Germany). Samples were collected within 1 h of the experimenter entering the barn according to Rettenbacher et al. [21], who found a first major peak 1 h after a stress pulse in laying hens. One dropping per bird was collected immediately after defecation, put into frost-resistant plastic bags and frozen on dry ice at a usual temperature of −78.5 ◦C. Droppings were transferred to a freezer after sampling. To determine baseline concentrations, pullets were sampled at 9:00 a.m., two days before transportation. For measurements of transportation and translocation stress, further droppings in both variants were collected 0 h, 3 h, 6 h, 10 h, 24 h, 48 h and 72 h after transportation. Initially, the flocks had been sampled 9 h and 12 h (instead of 10 h) after transportation. However, at these time points, only a few (if any) birds defecated. To prevent an unworkable additional work load and a possible violation of the numerical limit of permitted experimental birds, we decided to collect samples 10 h after transportation. Taking the circadian rhythm into account, flocks of Variant II were sampled additionally 34 h and 58 h after transportation. Altogether, 5751 droppings were collected and analyzed.

In the laboratory, 0.5 g of each sample was suspended in 5 mL of 60% (*v*/*v*) methanol by shaking for 30 min on a multi-vortex (RapidVap, Labconco, Kansas City, MO, USA) [22]. When a smaller portion had to be used, an aliquot of methanol was added. After centrifugation (GS-6KR Centrifuge, Beckman, Krefeld, Germany) for 15 min, aliquots of the supernatant were diluted 1:10 with assay buffer, and concentrations of fecal corticosterone metabolites (CM) were determined with a cortisone enzyme immunoassay [21]. The applied method has been validated physiologically and biologically for chickens by Rettenbacher et al. [21,23].

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

The level of fear of humans is an important determinant of welfare of pullets and laying hens. Regular handling of pullets is fear reducing [24], and positive additional human contact of laying hens reduces their fear level and influences their corticosterone level in blood positively [25]. In contrast, fear-inducing humans reduce the wellbeing of animals [26]. Accordingly, we tested each flock on avoidance and approach behavior by using the touch test of Raubek et al. [27] to assess the birds' reaction to an unfamiliar human. The test was performed with each flock by the same test person, who was unfamiliar to the flock before test. The test person wore protective clothing such as a blue overall, plastic overshoes and a hair cloth. Tests were carried out in the roofed outdoor run area (winter garden) of the rearing farm. Entering the winter garden was the initial contact between flock and test person. The unfamiliar test person moved slowly—one step per second—through the winter garden, approached a group of at least three pullets, squatted for 10 s and then counted all pullets within one arm's length around her. Thereafter, the test person tried to touch one bird after the other. The test was carried out until 33 groups were examined. Any attempt to approach a group or squat down was counted, even if all pullets retreated from the test person [28].
