Milk cortisol response to group relocation in lactating cows Nataliya Poscic, Gianfranco Gabai, Bruno Stefanon, Laura Da Dalt, Sandy Sgorlon Supplementary material Detailed materials and methods Animals and management Collection of milk samples for cortisol analysis was performed on commercial dairy farm in Northern Italy. The farm involved adheres to a high standard of animal care that includes providing cows with nutritious diets, healthy living conditions and good veterinary care on best practice manual under the supervision of the licensed veterinarian. All animals were clinically healthy, were in their first, second or third lactation and were kept under the same feeding and management conditions. The cows were housed in one free stall barn with cubicle design and automated milking parlour. Cows were milked twice a day, had free access to water and were fed ad libitum once a day at 7:30 am a total mixed ration (TMR) based on corn silage and formulated to cover nutrient requirements (INRA, 1989). To ensure that no dietary variations occurred during the time window of the study, the ration formulation and the offered amount were recorded using registrations of the TMR mixed feeder. Automatic individual animal identification and milk yield recording system allowed to record individual variations in daily milk yield during all the sampling period. Experimental design and sample collection The experiment was performed on Holstein Friesian (HF) and Norwegian Red (NR) cows and consisted of two studies (Supplementary Table S1), which were aimed at measuring the concentration of milk cortisol without perturbations and following the relocation of animals between production groups.