Atipamezole

Dogs were fasted a minimum of 12 h. Sample collection (phlebotomy) was performed in the morning. Basal blood sampling was performed 90 min after butorphanol (Fox et al., 1998 (link)), trazodone (Ashraf, Shariati & Zamanpor, 2014 ), and saline administration, with sampling performed 120 min after dexmedetomidine administration. Sampling intervals were based on projected time of maximal effect on basal cortisol based on limited prior data (Maze et al., 1991 (link); Restitutti et al., 2012 (link); Fox et al., 1998 (link); Ambrisko, Hikasa & Sato, 2005 (link); Morris et al., 2020 (link); Manthey et al., 2011 (link); Ashraf, Shariati & Zamanpor, 2014 ). When possible, phlebotomy to collect blood for hormone analysis was performed using the jugular vein. Following phlebotomy, dogs were administered cosyntropin (5 μg/kg, IV, Cortrosyn^TM; Amphastar Pharma, Rancho Cucamonga, CA, USA) via cephalic vein, followed by collection of a post-ACTH blood sample 1 h later (Carotenuto et al., 2019 (link); Guzmán Ramos et al., 2022 (link)). Blood was allowed to clot at room temperature and then centrifuged at 2,200 × g for 15 min to separate serum. Serum was stored at −20 °C pending sample analysis. After blood samples were collected, atipamezole (Antisedan; Zoetis Inc., Kalamazoo, MI, USA) was given at the clinician’s discretion to dogs that received dexmedetomidine.

All sampling was performed by a licensed veterinarian in the Sloth Sanctuary's veterinary clinic between September 2014 and November 2015. Between 30 and 50 hairs (including the follicular bulb) were collected from a total of 98 Hoffman's two‐fingered sloths (Choloepus hoffmanni) originating from numerous regions across Costa Rica (Table S1). All sloths sampled were born in the wild but were being maintained at the Sloth Sanctuary of Costa Rica for rehabilitation. The locations from which each sloth originated were obtained from sanctuary records. Hairs were collected from the lower back of each animal using tweezers, and all sloths were sampled under anaesthesia to minimize handling‐induced stress. Sampling took place during scheduled health checks, to avoid unnecessary sedation. 1 mg/kg of ketamine (Ketamina 50^®, Holliday Scott) and 0.008 mg/kg of dexmedetomidine (Dexdomitor^®, Zoetis) were administered intramuscularly by a licensed veterinarian following standardized sanctuary procedures. The anaesthesia was reversed using 0.008 mg/kg of anti‐sedative (atipamezole; Antisedan^®, Zoetis). The hair samples were exported to Swansea University for analysis.

Animals were placed under ketamine+dexmedetomidine anesthesia (100+0.5 mg kg⁻¹, Zoetis, Florham Park, NJ, USA) before surgery using a stereotax. For IL infusions, a double-barreled guide cannula (C235GS system, Plastics One, Roanoke, VA, USA) was implanted. The guides were placed using the left internal guide as reference, 1.8 mm anterior to the bregma, 0.5 mm lateral and 2.5 mm ventrally. For BLA infusions, two guide cannulae mounted on a base plate (C315GS system, Plastics One) were implanted. The guides were placed 1.25 mm posterior 3.3 mm bilateral to the bregma and. The guide and dummy cannulae projected 3 mm below the base plate. After surgery, mice were given subcutaneous injections of atipamezole (5.0 mg kg⁻¹, Zoetis) to reverse anesthesia and the non-steroidal anti-inflammatory meloxicam (1 mg kg⁻¹) as an analgesic. Mice recovered for at least 4 days before experimentation.