CORTISOL CONCENTRATIONS IN MALE ALASKAN MOOSE (Alces a. gigas) AFTER EXOGENOUS ACTH ADMINISTRATION George A. Bubenik1, Charles C. Schwartz1 and John Carnesl 1Department of Zoology, University of Guelph, Guelph, Ontario, Canada, Nl G 2Wl; 2Alaska Department ofFish & Game, Soldotna, Alaska 99669 USA. ABSTRACT: Blood levels of cortisol were determined in five yearling Alaskan moose after an exogenous administration of 40 I.U. of ACTH. A rapid elevation of cortisol concentration (over l5J.1 g/100 mL) within 60 min. of ACTH injection demonstrated an unexpectedly high level of adrenocortical response to a simulated stress. The results in moose are compared to several other deer species. ALCES VOL. 30 (1994) pp.65-69 Endocrine investigations of reindeer To expand our knowledge of stress adap­ (Rangifer tarandus tarandus) and moose tations of cervids and to compare these data to (Alces alces) are rare. Stress-induced varia­ other deer species, we examined the time tions of serum corticoid levels in moose were course of cortisol concentrations in Alaskan investigated by Franzmann and et al. (1975) moose after an exogenous administration of and in reindeer by Rehbinder and Edqvist ACTH. (1981) and Wiklund et al. (1993). Winter, summer and fall concentrations of MATERIAL AND METHODS ), ) triiodothyronine (T3 thyroxine T4 and Five tame, male, yearling Alaskan moose cortisol in moose were reported by Nilssen et (A. alces gigas) born and raised by their , al. (1985) and seasonal changes ofT3 T4 and mothers at the Kenai Moose Research Center growth hormone were published by Ryg (MRC) at Soldotna, Alaska, were tranquilized ( 1982). However, concentrations or seasonal with a 2:1 mixture of xylazine hydrochloride profiles of many other hormones in moose (Anased - Lloyd Lab. Shenandoah, Iowa, remain undetermined. USA) and ketamine hydrochloride (Ketaset - In several other cervids, the response to A veco Co. Fort Dodge, Iowa, USA), using stress has been quantified by measuring plasma pressurized darts shot from a blow pipe. The concentrations of cortisol after an exogenous dosages of the immobilizing mixture varied administration of the pituitary between 3 and 3.5 mglkg. The induction times adrenocorticotropin (ACTH)(Seal et al. 1982, were between 5 and 10 minutes. Once anaes­ SmithandBubenik 1990,Bubeniketa!.1991, thetised, a teflon cannula (Criticon, Cathlon, BubenikandBartos 1993). The time course of gauge 18 - Mississauga, Ontario, Canada) the elevation of cortisol in response to ACTH was implanted into the jugular vein and se­ may be used as an indicator of stress adapta­ cured with a suture. Moose were then tions which each species developed in re­ sequentially sampled (5 cern each time) for4 sponse to their particular environment hr. All experiments started between 0900 and (Bubenik and Reyes-Toledo 1994). Such 1000 hr. The first experiment [the treatment measurements were performed in a wide vari­ with 40 International Units (I.U.) of ACTH] ety of wild and domesticated mammals (Friend was performed on April 27, the second ex­ et al. 1977, Fulkerson and Jamieson 1982, periment (control with 0 I.U.) on May 18, Seal et al. 1982 ). 1993. The reverse order of dosages was used 65 CORTISOL IN MOOSE AFTER ACTH. - BUBENIK ET AL. ALCES VOL. 30 (1994) to minimise the effect of unfamiliar stress on All assays were performed in duplicates. The the baseline concentrations of cortisol. intra- and interassay coefficients of variance After the first 3 samples, taken 10 min were 5.2 and 13.4%; the recovery averaged apart into pre-heparinized syringes, either 0 93.5%. The sensitivity of the assay was found or 40 I. U. of porcine ACTH (ACTHAR Gel, to be less than 0.14 g/100 mL. Armour Pharmaceutical, Kankakee, Ill, USA), To measure cortisol response to ACTH, diluted in 5 ml of saline, were administered the area under the curve after ACTH (40 I. U .) intramuscularly (i.m.) After ACTH, three was compared with the response after 0 I.U. additional blood samples were taken 10 min. The cut-off points for the calculations was apart, two samples 30 min. apart and the final chosen at the 90 min interval, the period of two samples at 60 min intervals. During sam­ maximal cortisol elevation. pling, animals were maintained in a semi­ For statistical analysis we used SAS Gen­ conscious stage, mostly in a sternal recum­ eral Linear Models Procedure, where classes bence, by an infrequent administration of were ACTH doses, Treatment (Pre-treatment small doses (100 mg) of xylazine. In the vs Post-treatment), Time and Individual Ani­ previous study (Bubenik and Bartos 1993), mals. these maintenance doses did not influence the cortisol secretion in any statistically signifi­ RESULTS cant way. Details of the sampling procedure The model (Fig.1) proved significant vari­ were published in previous papers (Smith and ation F(17,79)=12.37, P<O.OOOl. All classes Bubenik 1990; Bubenik and Bartos, 1993). appeared influential. For ACTH doses F After completion of the sampling all moose (1,79)= 17.43, P<O.OOOl, for Treatment received 12 mg of yohimbine HCI and antibi­ F(1,79)=62.21, P<0.0001, for Time otics. F(6.79)=14.51, P<0.0001, and for Individual Blood was immediately centrifuged and Animal F(8,79)=2.03, P<0.06). plasma frozen until laboratory analysis of Cortisol values (Mean ± Standard Error) cortisol levels by a commercial RIA kit (Jot­ increased after time application in both groups dan Bioclinical Inc., Scarborough, Ontario). (for 0 I.U. from 2.96 ± 0.29 to 4.21 ± 0.44; 16 " 0~ 12 '..=.01 0 .. 8 :e0 (J - 40 l.U. ACTH -- 0 LU. ACTH o~~~~~~~~~~~~~~ -30 0 30 60 90 120 150 180 210 f Time (minutes) Fig. 1. Average cortisol levels(± S.E.) in plasma of five yearling moose sampled before and after ACfH administration (arrow). 66 1111!!!!' r ALCES VOL. 30 (1994) BUBENIK ET AL. -CORTISOL IN MOOSE .AFI'ER ACTH ,1:1 :I P<O.Ol; for40 I.U. from 6.06±0.78 to 12.57 concentrations (Bubenik et al. 1977), sea­ ± 0.19, P<0.0001). The cortisol concentra­ sonal variation of cortisol in white-tailed deer 1: tions rose rapidly within the first 60 minutes (Bubeniketa/.1975, 1983), pudu (Bubeniket and peak levels (15.4 and 15.6Jlg/100 mL), al.-unpublished data) or reindeer (Bubenik et were achieved 60 and 90 min, respectively, al. - unpublished data) was mostly non-sig­ after the injection of ACTH. While pre-treat­ nificant. However, Bubenik and Leatherland ment values did not differ significantly be­ (1984) observed significantly higher values tween the two groups (P<0.92), the post­ of cortisol during the rut in a group of white­ treatment values were markedly higher in the tailed bucks which behaviorally appeared 40 I.U. group when compared to 0 I.U. treat­ much calmer than a comparable group of ment(12.57 ±0.19vs4.21 ±0.44, P<0.0001). excitable males. Whereas the basal values of DISCUSSION glucocorticoids in cervids are relatively low, Elevation of cortisol concentrations dur­ compared to some other mammals (Brown et ing stress is a response essential for the sur­ al. 1971, Taylor et al. 1976) the ACTH­ vival of animals (McEwen et al. 1986). A induced elevations can reach over 20-30Jlg/ reproducible stress is difficult to administer; 100 mL. The relatively high pretreatment therefore simulated stress responses (by i.m. concentrations (approximately 6J.1g/100 ml) administration of pituitary ACTH) have been in our 40 I.U. experiment (Fig.l) was most investigated in various cervids such as rusa likely caused by stress due to confinement. It deer (Cervus rusa timorensis), roe deer appears that the young animals were not en­ (Capreolus capreolus), red deer (Cervus tirely habituated to their smaller holding pens elaphus ), fallow deer (Dama dama ), axis (Axis into which they were moved a few days before axis), white-tailed deer (Odocoileus the experiment. During the immobilization virginianus) and pudu (Pudu puda) (Bubenik (done by darts) they tried to escape and be­ et al. 1991, Bubenik and Bartos - 1993, came agitated. During the second experiment Bubenik and Reyes-Toledo 1993, Seal et al. (control) performed three weeks later, the 1982, Smith and Bubenik 1991, Sempere & moose were much calmer and their Bubenik - unpublished, Van Mourik and pretreatment levels (3-4Jlg/1 00 mL) were more Stelmasiak, 1984). To compare the stress re­ similar to the basal concentrations found in sponses among deer species and relate it to red deer, fallow deer (Bubenik and Bartos their respective habitats and behavior, boreal 1993), white-tailed deer (Smith and Bubenik cervids such as reindeer and moose should 1990) and reindeer (Rehbinder and Edquist also be investigated. Seasonal cortisol con­ 1981, Wiklund et al. 1993). centrations have not been determined in moose, The smallest increase of cortisol concen­ but were reported in reindeer. Plasma cortisol trations after ACTH was observed in small, concentrations in male reindeer did not differ solitary, reclusive deer, such as the roe-deer between summer and winter in one study (2.1Jlg/100 mL) (Sempere and Bubenik- un­ (Ringberg et al. 1978). In contrast, Nilssen et published data and the pudu (2.6J.1g/l00 mL) al. ( 1985) reported higher cortisol concentra­ (Bubenik and Reyes-Toledo 1994). These tions in summer than in fall and winter. In species escape their predators by hiding in a response to stress, cortisol concentrations rose thick forest understory. In gregarious deer, rapidly in reindeer (Rehbinder and Edqvist which outrun their predators, average levels 1981; Wiklund et al. 1993) as well as in the were higher: approximately 8Jlg/100mL in moose (Franzmann et al. 1975). fallow deer and 10J.1g/l00 mL in red deer Because of a large variation in individual (BubenikandBartos 1993) and l2Jlg/100mL 67 CORTISOL IN MOOSE AFfER ACTH.- BUBENIK ET AL.