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 to both ground (AUC_G) and to increase (AUC_I). The AUC_I increased markedly between C1 and C2 (P < 0.05) in ST bulls, and no effects were observed for AUC_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 < 0.01, ICC = 0.41) and lacked in AT bulls (AUC_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 < 0.001; ICC = 0.53; AUC_I: r = 0.58, P < 0.001, ICC = 0.52). Assignment to high and low responder groups based on peak cortisol concentration in C1 did not persist over time. H+ bulls showed higher AUC_I in C2 and C3 (P < 0.1 and P < 0.05, respectively) than H− bulls. The M group differed from the H− group only in C3 (P < 0.05). Thus, the effect of horn status on ACTH challenges needs further investigation. In conclusion, our results report poor repeatability of the cortisol response to ACTH challenges for challenges performed in different physiological states and moderate repeatability for challenges performed within the same physiological state. Moreover, they point out the importance of standardization of TOD when performing repeated ACTH challenge.

评估动物的压力或福利时，经常重复进行促肾上腺皮质激素（ACTH）攻击。据我们所知，牛中ACTH挑战的可重复性，尽管对这种类型的进一步研究至关重要，但尚未成为研究的主题。因此，本研究的目的是评估ACTH挑战在不同号角状态的育肥公牛中的可重复性。八十一头公牛连续3次受到ACTH挑战。第一个挑战（C1）在1.5 mo的犊牛中进行。随后，将动物定性为皮质醇高或低反应者，或者动物脱臼或留有角。然后将他们分配给3个饲养组中的1个：有角的组（H +），无预算的组（H-）和混合的组（M；有50％的有角和50％的无预算），每个组包含相等数量的高和低响应者。第二次ACTH攻击（C2）在11 mo岁时进行。挑战的一天中的时间（TOD）是固定的（ST =同一TOD）或在C1和C2之间交替（AT =替代TOD）。在与C2相同的TOD之后7天进行第三次ACTH攻击（C3）。每次静脉注射ACTH前60分钟和30分钟以及之后30、60、90、120和150分钟采集唾液样品。相对于两个地面（AUC）计算曲线下面积（AUC）每次静脉注射ACTH前60分钟和30分钟以及之后30、60、90、120和150分钟采集唾液样品。相对于两个地面（AUC）计算曲线下面积（AUC）每次静脉注射ACTH前60分钟和30分钟以及之后30、60、90、120和150分钟采集唾液样品。相对于两个地面（AUC）计算曲线下面积（AUC）_G）并增加（AUC _I）。ST公牛的C1和C2之间的AUC _I显着增加（P <0.05），ST公牛或AT公牛的C1和C2之间的AUC _G以及C2和C3之间的任何AUC均未观察到影响（P > 0.1）。ST公牛的AUC _G和AUC _I在C1和C2之间的总体可重复性分别反映了2种不同生理状态之间的可重复性（AUC _G：r = 0.24，P > 0.1，组内相关系数（ICC） ）= 0.21; AUC _I：r = 0.48，P  <0.01，ICC = 0.41）并且缺少AT多头（AUC_G：r = 0.07，P > 0.1；ICC = 0.03；AUC _I：r ＝ 0.08，P  ＞ 0.1，ICC ＝ 0.06）。C2和C3之间的AUC _G和AUC _I的重复性适中（AUC _G：r = 0.59，P <0.001; ICC = 0.53; AUC _I：r = 0.58，P <0.001 ，ICC = 0.52）。根据C1中皮质醇峰值浓度，高，低反应者组的分配不会随时间持续。H +公牛在C2和C3中显示较高的AUC _I（P <0.1和P分别<0.05）。M组仅在C3上与H-组不同（P <0.05）。因此，喇叭状态对ACTH挑战的影响需要进一步研究。总之，我们的结果报告了皮质醇对不同生理状态下进行的挑战对ACTH挑战的可重复性较差，而在相同生理状态下进行的挑战具有中等的可重复性。此外，他们指出了执行重复ACTH挑战时TOD标准化的重要性。