*2.1. Experimental Design*

The study was conducted with organically mixed-held pullets of two genetic lines—H&N Super Nick (HNS) and H&N Brown Nick (HNB)—of a commercial breeder and distributor (H&N International, Cuxhaven, Germany). Parent animals were imported to Switzerland and raised as organic laying hens. The experimental unit consisted of pullets and laying hens of 15 flocks, which were reared and kept according to the guidelines of Bio Suisse (Association of Swiss Organic Farming Organizations, Basel, Switzerland) [9] on free-range farms. Each rearing farm raised 4000 pullets, and farms of laying hens kept 2000 birds. The average ratio between HNS and HNB normally was 50:50 to 60:40. The transport to the farm of laying hens was realized at the age of 18 weeks. The study was based on two practically relevant commercial transport variants—with and without transportation break—which were categorized according to distance and length. Variant I "transport overnight" (transportation was performed with break) was compared with Variant II "direct transport" (transportation was performed without break). On average, 2014 birds were transferred on each transit. Each plastic crate (90.5 × 61.5 × 31.5 cm) was loaded with 16 pullets according to the Swiss Order on the Protection of Animals [4]. Because the start of loading also means the starting point of stress, we defined the time from the beginning of loading until the end of unloading as "time in plastic crate" or transport duration. Thus, the average transport duration was 13.5 h for Variant I and 5.0 h for Variant II, whereas the mean journey time alone was 2.6 h for Variant I and 1.0 h for Variant II (time on the road). Loading regularly began at 7 p.m. The legally prescribed transport duration was never exceeded [4]. We timed our investigation to include winter, spring, and summer.

Temperature was measured with HOBO U10 (temperature data loggers, Onset Computer Corporation, Bourne, MA, USA) inside of the stable at animal head height and during transportation inside of the plastic crates on the upper edge. For both transport variants, temperature was recorded during the whole investigation period every 10 min per flock. Means of minimum and maximum temperature values during the testing period (January until July) for Variant I ranged from 11.1 to 32.3 ◦C (rearing farm), 1.9 to 34.7 ◦C (transportation vehicle) and 3.8 to 34.1 ◦C (farm of laying hens) and for Variant II from 7.6 to 26.3 ◦C (rearing farm), −8.9 to 28.8 ◦C (transportation vehicle), and 6.1 to 26.2 ◦C (farm of laying hens).

Animal husbandry varied according to the individual farmer's management.
