**1. Introduction**

The World Organization for Animal Health provides a reference document [1] of international standards for animal health and zoonosis; it grants animals kept under human care the internationally recognized "five freedoms" of welfare described as follows: freedom from hunger, thirst and

malnutrition; freedom from fear and distress; freedom from physical and thermal discomfort; freedom from pain, injury and disease; freedom to express normal behavior patterns.

The Swiss consumer assumes that organic livestock farming applies more species-appropriate animal husbandry corresponding to the animals' natural living conditions, and thus provides better animal welfare than conventional farming practices [2]. Animal transport is an exceptional situation, because animal welfare may be compromised for a certain time. The European Commission Regulation (EC) No 1/2005 [3], limits the transport duration of poultry to less than 12 h. The Swiss Animal Welfare Regulation has limited the maximum duration of animal transport to less than 8 h since its amendment to Art. 152a (1) of 28 October 2015 [4], like the German Animal Welfare Transport Regulation [5]. Whereas some of the German organic labels further limit the maximum transport durations to less than 4 h or 2 h [6–8], none provide specific requirements for poultry, pullets, or laying hens. Similarly, the Swiss organic labels Bio Suisse [9] and KAG (Consumer-Working-Group; German designation: Konsumenten-Arbeits-Gruppe) [10] refer to the transportation guidelines of the Swiss Animal Protection [11], which are designed for big and small animals, not including economic fowl. All regulations, including EC No 1/2005, disregard loading and unloading time (Table 1).

Various stressors such as climate, environment, nutrition, physical, and social and physiological conditions are likely to influence welfare and performance [12]. The present study examined the effects of transportation and translocation on the stress hormone levels of 18-week-old organically reared pullets of two mixed-held layer lines—H&N Super Nick and H&N Brown Nick—in transit from the rearing farm to the farm of laying hens (novel environment) in Switzerland. The geographically dispersed locations of rearing and laying farms require that hens are transported on the road over distances of varying lengths. We compared two practice-oriented transport variants. Although the transportation process is supposed to be the most stressful environmental challenge experienced by broilers [13], catching and handling of the birds may already have profound effects on the degree of physiological stress, and it may reduce welfare during the upcoming transit [14–16]. Thus, we took into account the entire duration of the hens being held in plastic crates, including loading and unloading time.

To evaluate the stress response to the two transport variants, we measured corticosterone metabolites non-invasively in excretions of the pullets. Fecal corticosterone metabolites have previously been used as a reliable indicator of adrenocortical activity [17].

The project was initiated by Switzerland's largest organic egg supplier—hosberg AG in Rüti—and was supported by two other Swiss-based companies—Wüthrich Brüterei AG in Belp and Prodavi AG in Schötz. Participation in "LTK (Institute of Laboratory Animal Sciences) Module 2: Training for Persons Responsible for Directing Animal Experiments" was required as a prerequisite for the intercantonal approval by the Swiss Cantonal Veterinary Office in Zurich, and the project was approved on 6 January 2016, under registration number ZH196/15. The study refers to Europe, with special emphasis on Switzerland.


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**Table 1.** Comparison between Switzerland, Germany and Austria regarding maximum stocking densities of pullets and laying hens per pen, as well as minimum space requirements and maximum duration in the transit of pullets according to transnational (European Union, EU), federal, and label-specific regulations.

#### **2. Animals, Materials and Methods**
