The ACTH challenge and its repeatability in fattening bulls—influences of physiological state, challenge time standardization, and horn status

Abstract Adrenocorticotropic hormone (ACTH) challenges are frequently performed in repetition when evaluating stress or welfare in animals. To our knowledge, the repeatability of ACTH challenges in cattle, although fundamental to further studies of this type, has not yet been the subject of research. Therefore, the objective of this study was to evaluate the repeatability of ACTH challenges in fattening bulls of different horn status. Eight-one bulls were subjected to 3 consecutive ACTH challenges. The first challenge (C1) was performed in calves aged 1.5 mo. Subsequently, animals were characterized as high or low cortisol responders and either disbudded or left with horns. They were then assigned to 1 of 3 rearing groups: a horned group (H+), a disbudded group (H−), and a mixed group (M; 50% horned and 50% disbudded), with each group containing an equal number of high and low responders. The second ACTH challenge (C2) was performed at the age of 11 mo. Time of day (TOD) of challenge was either fixed (ST = same TOD) or alternated (AT = alternate TOD) between C1 and C2. The third ACTH challenge (C3) was performed 7 d after and at the same TOD as C2. Saliva samples were taken 60 and 30 min before and 30, 60, 90, 120, and 150 min after each intravenous ACTH injection. The area under the curve (AUC) was calculated with respect both to ground (AUC G ) and to increase (AUC I ). The AUC I increased markedly between C1 and C2 ( P G between C1 and C2 in ST or AT bulls, nor for any AUC between C2 and C3 ( P > 0.1). The overall repeatability of AUC G and AUC I between C1 and C2, reflecting the repeatability between 2 different physiological states, was poor and moderate, respectively, for ST bulls (AUC G : r = 0.24, P > 0.1, intraclass correlation coefficient (ICC) = 0.21; AUC I : r = 0.48, P G : r = 0.07, P > 0.1; ICC = 0.03; AUC I : r = 0.08, P  > 0.1, ICC = 0.06). The repeatability of AUC G and AUC I between C2 and C3, reflecting the repeatability within the same physiological state, was moderate (AUC G : r = 0.59, P I : r = 0.58, P I in C2 and C3 ( P P P