Glucocorticoid secretion from the adrenal cortex is stimulated by ACTH (adrenocorticotropic hormone) and is the endocrine system's first response to stressors. In the blood, ACTH rises within minutes of stress exposure, and is followed one to four hours later by a peak in glucocorticoid concentration.1,2,5-7 For this reason, glucocorticoids (hydrocortisone/cortisol in large mammals) and ACTH are utilized as indicators of stress in species. Knowledge of the physiologic and pathologic concentrations of these indicators is important because stress can disrupt metabolism and reproductive function and lead to immunosuppression or death.1,8
Physiologic and pathologic concentrations of cortisol and ACTH are not known for manatees. Health assessments of wild and captive manatees are typically completed within one hour's time. Therefore, if manatees are like other animals whose cortisol values peak more than one hour after stress induction, blood-based cortisol samples may not fully represent capture-associated stress. However, peak ACTH concentrations may be available from these same samples, making ACTH a better tool for diagnosing acute stress in manatees. High sensitivity assays for cortisol (hydrocortisone, Compound F) and ACTH (IMMULITE 1000®, Diagnostic Product Corporation, Los Angeles, CA) have been analytically validated in accordance with the Clinical Laboratory Improvement Amendment to measure these compounds in Florida manatees.
Blood samples were collected opportunistically during captures of wild manatees (n=83) or health assessments of long-term captive (n=15) and rehabilitating individuals (n=30). Blood was obtained via venipuncture from the brachial plexus and samples were acquired from manatees of both genders and a range of ages. The cortisol immunoassay was validated with both serum and lithium heparin plasma (lower limit of detection [LLOD] = 0.1 µg/dl) and the ACTH assay (LLOD = 10.0 pg/ml) utilized EDTA plasma.
For cortisol (serum and lithium heparin plasma), inter-assay and intra-assay coefficients of variation (CV) for precision were < 21% (CV < 30% required for cortisol immunoassays4). Linearity was assessed by dilution experiments, with adjusted R2 values > 0.87 for serum and > 0.96 for plasma. Methods were compared with serum and plasma cortisol radioimmunoassays (RIAs) at Cornell University's Animal Health Diagnostic Center and correlation coefficients (r) of 0.81 and 0.90 were observed, respectively. Reference intervals generated from serum cortisol results were established via ROC (Receiver Operating Curve) Analysis using known health and history information to distinguish among acutely stressed healthy, chronically stressed unhealthy, and unstressed healthy manatees. ROC Analysis indicated that cortisol concentrations < 0.2 µg/dl, > 0.2 µg/dl, and > 1.1 µg/dl are characteristic of unstressed healthy (n=26), acutely stressed healthy (n=74), and chronically stressed unhealthy manatees (n=28), respectively.
ACTH inter-assay and intra-assay CV were <9% (<10% required4). Linearity, assessed by dilution experiments, resulted in adjusted R2 values > 0.99. Preliminary ROC Analysis of acutely stressed healthy (n=15) and unstressed healthy manatees (n=6) suggests that acute stress is diagnosed by ACTH > 90 pg/ml. The highest ACTH value observed (643 pg/ml) occurred in a healthy manatee followed for 50 minutes prior to successful capture. Lower values occurred in healthy manatees (n=14) captured in a shorter time (90-364 pg/ml). ACTH in sick captive or rehabilitating manatees (n=13) ranged from 23.5-197 pg/ml. Long-term captive manatees assessed as unstressed and healthy (n=6) had ACTH concentrations ranging from 42-75.7 pg/ml.
Development of these assays has furthered understanding of normal physiologic and stress-related concentrations of cortisol and ACTH in manatees. The cortisol data obtained in this study for unstressed healthy manatees is in agreement with that obtained by Manire et al.3, where long-term captive manatees had mean serum cortisol concentrations of ~0.2 µg/dl. To our knowledge, no other studies have defined reference intervals for cortisol associated with varying levels of manatee health and stress, as we have. We have also described ACTH concentrations in manatees for the first time and defined an acute stress ACTH threshold for the manatee. Our findings of increased ACTH values accompanied by cortisol values near baseline (0.1 µg/dl) suggest that the cortisol increase in the manatee is delayed at least one hour after stress induction. For this reason, ACTH is a better indicator of acute stress in the manatee, given the current capture and restraint protocols. However, the validated cortisol assay provides a useful tool for examining chronic stress in the Florida manatee.
Many thanks to FWRI, Lowry Park Zoo, Miami Seaquarium, Mote Marine Lab, Sea World of Florida, and the USGS Sirenia Project for providing samples, and to Melanie Pate and Jim Burrow for excellent assistance in the laboratory.
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