Evaluation of adrenal function in serum and feces of Steller sea lions ( Eumetopias jubatus): influences of molt, gender, sample storage, and age on glucocorticoid metabolism

GENERAL AND COMPARATIVE

ENDOCRINOLOGY

www.elsevier.com/locate/ygcen

General and Comparative Endocrinology 136 (2004) 371–381

Evaluation of adrenal function in serum and feces of Steller sealions (Eumetopias jubatus): influences of molt, gender, sample

storage, and age on glucocorticoid metabolism

Kendall L. Mashburn and Shannon Atkinson*

University of Alaska, Fairbanks and Alaska SeaLife Center, P.O. Box 1329, 301 Railway Avenue, Seward, AL 99664, USA

Received 15 September 2003; revised 26 January 2004; accepted 28 January 2004

Abstract

Fecal corticosterone concentrations, measured via radioimmunoassay (RIA), were validated as a method to monitor adrenal

function in Steller sea lion physiology. Quantification of adrenal response to an acute stressor and relevance of data produced by

developed methodologies was determined through physiological challenge with exogenous administration of adrenocorticotropic

hormone (ACTH) to captive adult, reproductively intact, Steller sea lions of both sexes (n ¼ 3, 1 male, 2 female) during seasonal

molt. Following ACTH administration, serial blood and fecal samples were collected and analyzed by RIA to determine adrenal

response. Storage regimens and weather exposure were examined to establish external impact on fecal corticosterone concentrations.

High-pressure liquid chromatography (HPLC) of both serum and feces of Steller sea lions was employed to explore potential

gender-based differences extant in either sample media. ACTH challenges produced >3-fold increases in serum cortisol concen-

trations which were reflected in >18-fold increases in fecal corticosterone concentrations post-injection at 3.25 and 32 h, respectively,

and fecal corticosterone concentrations returned to baseline 52 h post-injection. Neither outdoor exposure to weather nor variation

in duration and temperature of freezer storage impacted fecal corticosterone concentrations. HPLC of individual fecal samples

produced eluate immunoreactivity profiles that differed consistently with both sex and age class. Techniques developed herein ef-

fectively detected physiologically relevant corticosterone data in Steller sea lion feces, unaffected by conditions likely to be en-

countered with field collection samples. Additionally, results quantify an acute response to ACTH and provide methodology for

examining chronically heightened adrenal activity in Steller sea lions.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Eumetopias jubatus; Steller sea lion; Cortisol; Corticosterone; Fecal glucocorticoids; ACTH; Adrenal glands; Hormones; Stress

1. Introduction

Since about 1970, the Western stock of Steller sea

lions have declined by an estimated 80% across the

geographic range from Cape Suckling, Alaska to the Seaof Okhostk, Japan (Braham et al., 1980; Merrick et al.,

1987). To date, there is no single identified cause of the

decline in endangered populations of Steller sea lions.

The bulk of previous research activities have addressed

diet, population trends, and metabolic demands (Mer-

rick et al., 1997). Most reproductive studies have been

* Corresponding author. Fax: 1-907-224-6320.

E-mail address: [email protected] (S.Atkinson).

0016-6480/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2004.01.016

based on observation or gross morphology of animals

harvested in the 1970s, 1980s, and 1990s (Ishinazaka

and Endo, 1999; Pitcher and Calkins, 1981; Pitcher

et al., 1998; Ruam-Suryan et al., 2002). Studies that

address the physiology of Steller sea lions are few andlimited to basic blood chemistries or related to fasting

and lipid metabolism (Bergman and Rea, 2000; Bishop

and Morado, 1995; Castellini et al., 1993; Rea et al.,

1998; Zenteno-Savin et al., 1997). The endocrine system

is the biochemical means of internal homeostatic regu-

lation, therefore, lack of knowledge in this area hampers

the investigation of factors suspected to impair Steller

sea lion physiology. Disruption or alteration in ‘‘nor-mal’’ endocrine patterns can create physiological im-

balances that impair the growth, reproduction, and

372 K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381

overall health of the sea lions. Detection of these im-balances can more clearly define goals for mitigation or

intervention.

Endocrine signals that indicate deviations from ho-

meostatic balance, or stress, include increased adrenal

output of corticoid hormones (Carrasco and Van de Kar,

2003; Mostl and Palme, 2002; Oki and Atkinson, in

press). Circulatory, salivary, or urinary concentrations

of glucocorticoids, and primarily cortisol or corticoste-rone, are often measured as indicators of stress. The

measurement of elevated glucocorticoids in the blood or

feces indicates that an animal has been exposed to a

stressor, but does not necessarily mean that an animal is

currently stressed. However, chronic elevation of these

hormones above an established baseline is often used as a

diagnostic tool to indicate an individual or population

that is compromised or may be at risk. Sampling forcorticosteroid diagnostics typically occurs in a longitu-

dinal fashion to account for normal circadian rhythms of

corticosteroids and the potential temporal response to

stress caused by collection procedures (Barriga et al.,

2001; Feldman et al., 1978; Walker et al., 2001). Blood,

saliva, or urine collection is not often practical in free-

ranging, non-domestic species due to logistical difficul-

ties and/or stress responses associated with capture orrestraint of animals. Sample collection from marine

mammals is often compounded by the inability to sample

any but the very young, physiologically compromised

(i.e., stranded or entangled), or well trained captive an-

imals. Non-invasive measurement of corticosterone in

feces from free-ranging animals has proven to be a

valuable tool for identification of stress responses in a

variety of species. This method also provides the capa-bility for longitudinal data collection such that stress

responses can be further defined as acute or chronic

(Cavagelli, 1999; Creel et al., 1997; Goymann et al., 1999;

Harper and Austad, 2000; Monfort et al., 1998; Terio

et al., 1999; Turner et al., 2002; Wasser et al., 2000). Once

validated for use in Steller sea lions, measurement of

fecal corticosterone will provide a viable alternative to

blood collection for the determination of adrenal outputand physiological stress in free-ranging animals.

The primary objective of this study was to determine

physiologic relevance of fecal corticosterone measure-

ments as indicators of stress or well-being in Steller sea

lions through detection of adrenal response to exoge-

nously administered adrenocorticotropic hormone

(ACTH). Second, as anesthesia was required for a pe-

riod of 3 h post-ACTH administration, it was necessaryto ensure that restraint and anesthesia did not contrib-

ute to measured adrenal response in either serum or

feces of challenged animals. The geographic distribution

of Steller sea lion rookeries requires that sample col-

lection occur during extended cruises with multiple sites

visited. As a consequence, both storage duration and

temperature vary. Thus, an additional priority for future

field applications was identification of environmental orstorage factors associated with field sample collection as

potential sources for data error as has been seen in other

species (Kahn et al., 2002; Washburn and Millspaugh,

2002). Finally, we endeavored to determine if detectable

gender differences existed in fecal corticosteroid ana-

lytes. Key Steller sea lion sample collection sites (rook-

eries) are typically populated with breeding, territorial

males, breeding females, and breeding females withpups. There is a paucity of data describing reproductive

endocrinology in free-ranging Steller sea lions in the

absence of physical manipulation of animals for sample

collection. Consequently, little information is available

representative of adrenal activity in typical territorial

behavior, reproduction or lactation in this species. In the

absence of these data, sex determination of collected

scats eliminates gender-bias in endocrine analysis of fieldsamples at the population level.

2. Methods

2.1. Animals

Samples for in situ sample exposure, sample storage,anesthesia, and ACTH challenge trials were obtained

from three adult (1 male, 539 kg; 2 females, 200 kg each)

reproductively intact, captive Steller sea lions housed

under ambient conditions at the Alaska SeaLife Center

(ASLC, Seward, Alaska; N 60�120 latitude, W 149�420longitude). Free-range fecal samples for HPLC deter-

mination of sex and age class differences were collected

from Natoa Island (N 59�360 latitude, W 149�360 lon-gitude) and Chiswell Island (N 59�360 latitude, W

149�340 longitude). Age and sex data were obtained

through the use of remote cameras located on each is-

land. Samples were also collected from one female

Steller sea lion pup in the rehabilitation program at the

Alaska SeaLife Center.

2.2. Blood samples

All sera used for this study, whether derived from

whole blood collected via caudal plexus veinipuncture

(Experiment 1, Effects of isoflurane anesthesia) or

through hindflipper vein catheterization (Experiment 5,

ACTH challenge), were collected into serum separator

Vacutainers (Becton–Dickinson, Franklin Lakes, NJ).

All blood samples were refrigerated prior to serumharvest into 1ml aliquots and stored frozen at )80 �Cuntil radioimmunoassay (RIA).

2.3. Fecal sample extraction and preparation

Feces from individual Steller sea lions were fully

mixed, aliquoted (�5 g) and loaded onto a rotary

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381 373

evaporator (Speed-Vac Plus, SC110A; Savant Instru-ments, Holbrook, NY) and dried without heat. Dried

fecal samples were crushed into powder, after which

0.025–0.03 g was weighed and extracted as previously

described by Monfort et al. (1998). Methanol (MeOH)

extractant (100 ll) was aliquoted into polypropylene

tubes, dried under forced air, reconstituted in 400 llbuffer for a final 1:4 dilution. Sample dilutions were

stored frozen at )20 �C until RIA.

2.4. Cortisol RIA

A solid-phase RIA kit (Diagnostic Products, Los

Angeles, CA) for cortisol was validated for use with

unextracted, undiluted Steller sea lion serum. Radio-

activity of bound portion was determined using a

gamma counter (Gamma C12, Diagnostic Products,Los Angeles, CA). All samples from a single experi-

ment were analyzed in duplicate within the same assay

to reduce variation. The RIAs were performed per

manufacturer instructions with the exception that all

volumes were halved and an additional standard was

added to the curve (i.e., one half the lowest standard)

to increase sensitivity. Manufacturer cross-reactivity

data are as follows: prednisolone (76.0%), 11-deoxy-cortisol (11.4%), prednisone (2.3%), cortisone (0.98%),

corticosterone (0.94%), tetrahydrocortisol (0.34%), 11-

deoxycorticosterone (0.26%), aldosterone (0.03%),

progesterone and pregnenolone (0.02%), flumethasone

(0.017%), and 0.01% or below for all other steroids

tested.

Serial dilutions of serum pools from both male and

female Steller sea lions (neat to 1:16) yielded displace-ment parallel to that of the cortisol standard curve.

Recovery of added cortisol (5–500 ng/ml) was 99.35%

(SD¼ 8.37; CV¼ 8.43%) for males (y ¼ 0:474þ 0:85x,r2 ¼ 0:99) and 92.05% (SD¼ 10.13, CV¼ 11.00%) for

females (y ¼ �0:103þ 0:98x, r2 ¼ 1:00). Interassay co-

efficients of variation for two separate assay controls

were 12.92 and 11.04% (n ¼ 4 for all samples assayed).

Intrassay coefficients of variation were <5% and assaysensitivity was 5.8 ng/ml.

2.5. Corticosterone RIA

A double antibody RIA kit (ICN Biomedicals, Ata-

scadero, CA) for corticosterone was validated for use

with extracted feces. All fecal samples were initially

analyzed at a dilution of 1:4 in diluent buffer providedby the manufacturer. Values from the RIA were cor-

rected for dilution, extraction efficiency, weight of fecal

material extracted, and expressed as ng/g dry weight.

The RIAs were performed according to manufacturer�sinstructions with the exception that all volumes were

halved and an additional standard was added to the

curve (i.e., one-half the lowest standard) to increase

sensitivity. Manufacturer cross-reactivity with othersteroids were: desoxycorticosterone (0.34%), testoster-

one (0.10%), cortisol (0.05%), aldosterone (0.03%),

progesterone (0.02%), and less than 0.01% for all other

steroids tested.

Serial dilutions of fecal extract for both male and

female Steller sea lions (1:1–1:28) yielded displacement

parallel to the standard curve. Recovery of corticoste-

rone added to fecal sample pools (range 12.5–500 ng/ml)was 90.3% (SD¼ 6.1; CV¼ 6.7%) for males (y ¼ 1:95þ0:88x; R2 ¼ 0:99) and 93.3% (SD¼ 15.2; CV¼ 16.4%)

for females (y ¼ 1:85þ 0:80x; R2 ¼ 0:99). Interassay

coefficients of variation for two separate assay controls

were 5.12 and 10.95% (n ¼ 9 for all samples). Intrassay

coefficients of variation were <5% and assay sensitivity

was 13.7 ng/ml.

2.6. High-pressure liquid chromatography

HPLC (Varian ProStar 210/215, Varian, Walnut

Creek, CA) was used to determine immunoreactive

corticosteroid components in Steller sea lion serum and

feces in the validated RIAs. Randomly selected fecal

extracts and unextracted sera were used to create 3ml

pools of each sex and sample type. The 3ml fecal extractpools were dried under forced air, rinsed with MeOH,

and re-dried twice to ensure that the majority of the pool

was washed to the bottom of the tube. Dried fecal pools

were reconstituted in 500 ll phosphate-buffered saline

(PBS) buffer (pH¼ 5.0). Similarly, 500 ll PBS was added

to serum pools and all samples were vortexed for 2min.

All pools were analyzed and fractions collected via

HPLC using a previously described method (Monfortet al., 1998). HPLC for individual animals was identical

to that of the pooled samples, with the exception that

larger portions (0.3–0.4 g) of dried feces from individuals

were extracted to ensure a high concentration of im-

munoreactive components. Resultant immunoreactivity

and radioactivity profiles were then obtained from

analysis of collected eluates and expressed per 1ml

fraction collected.

2.7. Experiment 1. Effects of isoflurane anesthesia

Animals were required to undergo anesthesia the first

3–4 h of an ACTH trial (Experiment 5), therefore, it was

necessary to determine the effects of isoflurane (Isoflu-

rane, USP; Halocarbon Industries, River Edge, NJ)

anesthesia (delivered via Vet Tech Model LAVC-2000,JD Medical Distributing Company, Phoenix, AZ). Ad-

ditional sera collected from both male (n ¼ 1) and fe-

male (n ¼ 2) captive Steller sea lions during routine

veterinary care procedures was analyzed. Samples cho-

sen for the experiment were paired when possible, i.e.,

voluntary or restraint bleeds followed by anesthesia and

additional sampling blood sampling under anesthesia

374 K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381

usually occurred at varying times post-anesthesia so aneffort was made to use those samples obtained between

30 and 40min post-anesthesia. Voluntary samples or

restraint samples were collected between 20 and 30min

after positioning animals in trained ‘‘stationing’’ pos-

tures. Resultant serum concentrations were divided into

three categories, voluntary (n ¼ 8), restraint (n ¼ 7), or

anesthetized (n ¼ 19) and compared statistically be-

tween sexes and treatments.

2.8. Experiment 2. Change in fecal corticosterone con-

centration due to freezing regimen

To assess the impact of storage variation, freshly

voided scat from a male and two pooled fresh scats from

two female Steller sea lions were fully homogenized and

subsampled. Three subsamples, for each sex were im-mediately dried (within 30min post-defecation¼Time

0) on the rotary evaporator and placed in a )20 �Cfreezer until processing and RIA. Thirty subsamples for

each sex (5–6 g wet weight) were placed into tubes, split

into two groups, and stored at either )20 or )80 �C(n ¼ 15 each temperature). Triplicate subsamples for

each sex were then removed weekly from Time 0 for 5

weeks from each freezer, dried without heat, crushed,and stored frozen at )20 �C until RIA. At the end of 5

weeks, all samples (total samples ¼ 66) were processed

then assayed in duplicate to eliminate handling or in-

terassay variation. Resultant fecal corticosterone con-

centrations and freezing regimen were statistically

compared to Time 0 for each sex.

2.9. Experiment 3. Effects of in situ exposure on fecal

corticosterone concentrations

To assess the effects of prolonged weather exposure

on fecal corticosterone, a freshly voided scat from a

male and two pooled fresh scats from two female Steller

sea lions were fully homogenized and subsampled.

(n ¼ 3, Time 0). The remainder was left outdoors, un-

covered, from between June 15 to July 05, 2001, whichcorresponds to typical field collection dates. Exposed

scat was subsampled in triplicate at 1, 2, 3, 4, 5, 6, 8, 10,

12, 18, 24, 36, 48, 60, 72, 96, and 108 h (at which time

scat was completely desiccated) post-defecation. All

subsamples were frozen at )20 �C until concurrent

analysis. Resultant fecal corticosterone concentrations

for each sex and freezing regimen were then statistically

compared to Time 0 for each sex.

2.10. Experiment 4. Differences in captive vs. free-

ranging, sex, and age class fecal immunoreactivity via

RIA of HPLC fractions

To assess similarity of captive and free-range ani-

mals and differences between sex and age class with

regards to fecal corticosterone concentrations, immu-noreactive components of free-range Steller sea lion

scat extracts were separated using HPLC and identified

for comparison with captives via the corticosterone

RIA. Feces of free-range, known sex and age animals

from Chiswell and Natoa Islands, verified through use

of remotely controlled video cameras located on each

island, were collected, processed, then analyzed via

HPLC. Resultant fractions for individuals were ana-lyzed via RIA and immunoreactive corticosterone peak

profiles of free-ranging animals were then compared to

those of captive animals housed at the Alaska SeaLife

Center.

2.11. Experiment 5. ACTH challenge

Adult, reproductively intact Steller sea lions (1 male,2 female) undergoing seasonal molt were treated with a

single injection (2 IU/kg, I.M.) of ACTH in a long-act-

ing gel preparation (synthesized by Hadfield�s Phar-

macy, Edmonds WA). Feces voided from each female,

obtained immediately prior to injection, was considered

Time 0. Time 0 fecal material from the male was col-

lected via the housing for rectal temperature monitor. A

blood sample was drawn from the caudal plexus im-mediately prior to ACTH injection (Time 0) which al-

lowed animals to serve as their own controls. Female sea

lions, due to their smaller size and relative ease of

management were held in an open squeeze cage for 1 h

post-injection followed by 3 h of isoflurane anesthesia.

Blood samples (20ml) were drawn every 15min via an

indwelling catheter (18 gauge, 3 in.) placed in a hind-

flipper vein. The catheter was flushed with approxi-mately 3ml sterile heparinized saline (concentration:

25 IU/ml) after each blood draw. At each new blood

draw, the initial saline portion was discarded. The male

was anesthetized immediately post-injection for 3 h,

during which blood samples were collected as described

above.

After 3 h, animals were removed from isoflourane.

Once fully alert, females were transferred to a largerstainless steel cage (45 in. W� 50 in. H� 10L) with a

slightly sloped floor fitted with gutters on one side to

prevent admixing of urine and feces. The cage was

placed in a sheltered outdoor area and fitted with an

observation camera. Females were then each monitored

constantly for 91.5 h post-ACTH administration for a

total of 96 h. Behaviors were recorded throughout this

time and each defecation was homogenized and sub-sampled. Due to the male�s large size and intractability

in close confinement, a video-monitored dry-run was

utilized during the day until defecation; after defecation

he was in a monitored outdoor pool enclosure. Defe-

cations in the outdoor pool enclosure accumulated until

approximately 20:00 h, at which time they were col-

lected. The pool was subsequently drained each morning

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381 375

with defecations produced overnight mixed together andsubsampled. All fecal samples were stored at )20 �Cuntil processing and assay.

2.12. Statistical analysis

Data are presented as means� SEM unless otherwise

noted. Comparisons of pre- and post-treatment data

were analyzed using repeated measures analysis withSigmaPlot 2002, v.8.0 (SPSS, Chicago, IL).

Fig. 1. Immunoreactivity (–d–) in pooled serum (A) from male and (B) femal

before HPLC as reference. All units are expressed per 1ml fraction.

3. Results

3.1. HPLC (RIA validation and gender-specific profiles)

Results for HPLC of serum pools for both sexes

demonstrated that serum immunoreactivity (100%) co-

eluted with added tritiated cortisol but not corticoste-

rone tracer (Fig. 1). Both male and female fecal pools

exhibited an immunoreactive peak that co-eluted withthe added tritiated corticosterone but not cortisol

e Steller sea lions [3H]cortisol and [3H]corticosterone (–s–) were added

Fig. 2. Immunoreactivity (–r–) in pooled feces (A) from male and (B) female (B) Steller sea lions [3H]cortisol and [3H]corticosterone (–}–) were

added before HPLC as reference. All units are expressed per 1ml fraction.

Table 1

Summary of serum cortisol concentrations (ng/ml; mean� SEM) in

captive Steller sea lions undergoing blood sampling under voluntary,

restraint, and anesthetized conditions

Sex Voluntary Restraint Anesthesia P

Male 94.1� 2.8 N/A� 73.2� 4.8 0.08

n 6 10

Female 82.0� 9.4 73.0� 6.2 81.2� 13.1 0.603

n 3 7 9

Between

sexes P

0.147 N/A�� 0.558

*No samples available for analysis.**Unable to compare two groups due to lack of sample.

376 K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381

(Fig. 2). In addition, both fecal pools had an immuno-

reactive peak that eluted in the more polar regions of theelution gradient, but consistently differed in location

with sex.

3.2. Experiment 1. Effects of isoflurane anesthesia

Cortisol concentrations of serum samples before

or after anesthesia administration or whether taken

under restraint or voluntary conditions indicatedthat there was no difference between males and fe-

males (P ¼ 0:556, P ¼ N=A, P ¼ 0:14, respectively,

Table 1).

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381 377

3.3. Experiment 2. Change in fecal corticosterone con-

centration due to freezing regimen

Fecal corticosterone concentrations in triplicate

subsamples of the same scat did not differ between Time

0 and those frozen at )20 or )80 �C for up to five weeks

for either male or female samples. Mean concentration

values for male scat were 60.17� 16.81 ng/g dry weight

at Time 0, 46.28� 4.99 at )20 �C (P ¼ 0:522), and50.85� 5.17 at )80 �C (P ¼ 0:687) at the end of five

weeks. Mean concentration values for female scat were

97.06� 6.22 ng/g dry weight at Time 0, 82.37� 6.89 at

)20 �C (P ¼ 0:140), and 92.47� 9.41 at )80 �C(P ¼ 0:517) at the end of five weeks.

3.4. Experiment 3. Effects of in situ exposure on fecal

corticosterone concentrations

Triplicate subsamples of both male and female scat

left exposed to ambient conditions for up to five days

exhibited fecal corticosterone concentrations no differ-

ent than those measured in the same scat at Time 0.

Mean concentration values for male scat were

50.13� 5.72 ng/g dry weight at Time 0 and 45.11� 8.32

(P ¼ 0:311) at the end of five days. Mean concentrationvalues for female scat were 212.78� 17.36 ng/g dry

weight at Time 0 and 196.93� 13.08 (P ¼ 0:795) at theend of five days.

3.5. Experiment 4. Differences in captive vs. free-ranging,

sex, and age/class fecal corticosterone immunoreactivity

via corticosteroid RIA of HPLC fractions

As is indicated by the HPLC profiles, there was no

difference between relative positions of immunoreactive

corticosterone peaks in free-ranging and captive animals

(Fig. 3). Although adult animals exhibited differences in

immunoreactivity in the more polar regions of the elu-

tion gradient, pups of different genders did not. In

contrast, juvenile males presented HPLC profiles with

both immunoreactive peaks in the more polar regions ofthe elution gradient that had previously occurred in

association with a specific gender in adults tested.

3.6. Experiment 5. ACTH challenge

Serum samples were averaged for each collection time

for all animals (Fig. 4). To account for variation in

defecation times, fecal samples were grouped, and av-eraged according to 4-h blocks. Serum cortisol concen-

trations increased 2-fold within 60min with a >3-fold

peak at 195min (65.3–220.0 ng/ml) and were reflected in

peak fecal corticosterone concentrations 18-fold above

baseline (88.5–1605.3 ng/g dry weight) 32 h post-ACTH

injection (beginning at 23.5 h with a return to baseline at

48.5 h). There were no gender-specific differences in

timing, duration or fecal corticosterone concentrationsassociated with the adrenal response to ACTH admin-

istration.

4. Discussion

In this study, the ACTH challenge was used to sim-

ulate an acute stress on Steller sea lions and evaluate theanimal�s adrenal response. The results demonstrate that,

as an acute stressor, ACTH initiates activity at the level

of the adrenal glands by 30min post-ACTH injection.

The adrenal response was clearly reflected in a fecal

corticosterone peak 32 h following the stimulus and was

cleared within 52 h, indicating that the feces integrated

and cleared the signal. Quantification of the response to

a known acute stressor enables a definition for the du-ration and maximal amplitude of adrenal activity in the

feces and provides a benchmark to which responses to

other stimuli can be compared. Such a benchmark al-

lows for the comparison of fecal corticosterone con-

centrations to be used to evaluate physiological function

of Steller sea lions in response to a variety of potential

stressors.

Validation of commercial RIA methodology, whenmodified as described herein, detected increased corti-

costeroids released in response to exogenous adminis-

tration of ACTH. Analogous HPLC elution profiles in

free-ranging and captive fecal samples indicate that the

extraction/RIA is an effective tool for the assessment of

adrenal activity in both wild and captive Steller sea li-

ons. The temporal relationship of serum cortisol to fecal

corticosteroids established through the ACTH challengedata enables appropriate protocols such that the timing

of the collection does not interfere with fecal cortico-

sterone concentrations. Further, knowledge of the

‘‘time-lag’’ relationship of serum to fecal corticosterone

allows extrapolation from collection dates to determine

relative contribution of potential stressors (e.g., weather

or entanglement) to corticosterone concentrations in

fecal samples.Outdoor exposure and freezer storage variability did

not significantly alter concentrations of corticosteroids

measured in the RIA utilized. From a field perspective,

this increases the number of samples that can be col-

lected from rookery or haul out sites by not limiting

collections for corticosteroid analysis to only freshly

voided scats. There is also increased confidence in the

comparison of scats from different research cruises withvariable storage temperature and duration.

The ability to determine the sex of a collected scat can

be of significant importance. The current means of scat

collection for adult Steller sea lions is typically on-site

collection without the presence of animals. Research

teams are rarely able to identify scats from individuals

collected from rookery or haul-out sites. Historically,

Fig. 3. Fecal corticosterone concentrations from captive (–d–) and free-ranging (–s–) Steller sea lion immunoreactivity HPLC profiles. (A) Captive

and free-ranging male scat. (B) Captive and free-ranging female scat. (C) Captive female and free-ranging male pup. (D) Captive female and free-

ranging female pup. Pups exhibited no difference in immunoreactive retention times between one another or those of a captive adult female. Fecal

samples from free-ranging juveniles (E) animal ID CIB4 and (F) animal ID CIB1 exhibited immunoreactive peak retention times that combined both

‘‘male’’ and ‘‘female’’ immunoreactive peaks in addition to those peaks co-eluting with radiolabeled glucocorticoids.

378 K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381

the primary focus of scat analysis has been for diet

composition and the assumption has been that both

sexes ingest the same prey and that males contribute

little, if any, fecal material for analysis from rookeries(Sinclair and Zeppelin, 2002). Sex and age-based differ-

ences occurred consistently using the methodologies

developed and warrents continued trials as known-sex/

age, free-ranging, Steller sea lion scats become available

from the field. Initial results of this study indicate that

the HPLC methodology presented herein may provide ameans to determine gender-based diet variation, which

is critical to the elimination of potential gender bias in

Fig. 4. Results of exogenous administration of ACTH in serum (–s–) and feces (–d–) of Steller sea lions (n ¼ 3). A peak increase of cortisol in serum

at 195min was reflected in a peak increase in fecal corticoids within 32 h post-ACTH injection.

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381 379

data analysis. Additionally, these methods appear to

successfully differentiate age classes. As reduced juvenilesurvival is suspected to be a major contributor to the

Steller sea lion decline (York, 1994), increased infor-

mation regarding diet and physiology in this age class is

essential.

Quantification of an acute adrenal response to a

stressor deductively allows for the definition of chronic

adrenal activity. While this study was not designed to

measure chronic stress in Steller sea lions, we can as-sume that repetitively high concentrations of fecal cor-

ticosterone from a single animal or population over time

may indicate the presence of a chronic stressor. The role

of stress in the declining Western stock of the Steller sea

lion is uncertain. Chronic stress has been shown to in-

terfere with numerous physiological processes critical to

individual survival, including immune function, metab-

olism, and disease resistance (Danzer and Mormede,1995; Larsen et al., 1998; St. Aubin and Dierauf, 2001).

Reproduction is negatively affected by chronic stress, as

is fetal growth and development, and has serious im-

plications at the population level should a significant

proportion of the breeding population be exposed (Le-sage et al., 2001; Rivier and Rivest, 1991). Evidence

pointing to treatment of each Steller sea lion rookery

site as a discreet foraging unit in the Western population

is mounting and includes reports of high site fidelity

(Sinclair and Zeppelin, 2002), variable degrees of re-

productive success between even adjacent rookeries (as

evidenced through live pup counts), and low rates of

exchange (Ruam-Suryan et al., 2002). Non-invasive,longitudinal examination of fecal corticosterone from

Steller sea lions at individual rookeries will allow iden-

tification of sites where animals are experiencing chronic

stress. This ability permits a more productive allocation

of resources to identify sources of chronic stressors and

in turn, enhances the knowledge base that drives man-

agement decisions.

While this technique has immediate applications forthe assessment of well-being in permanently captive,

transiently housed, or rehabilitation animals, additional

study is required before inferences can be made about

380 K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 136 (2004) 371–381

stress in Steller sea lions from unknown samples col-lected in the field. Future investigations to confirm the

presence of gender-specific corticosterone metabolites

and quantify the relative contribution of those identified

to total corticosterone concentrations are needed. Ad-

ditionally, the relationship between normal fecal corti-

costerone concentrations and the onset of parturition

and lactation in females, as well as territorial behavior

and breeding in males must be determined. Fecal sam-ples are typically collected when reproduction and ma-

ternal care are at their height and increased adrenal

activity that influence these events has been well docu-

mented in several mammalian species (Challis et al.,

2000; Whittle et al., 2001). Furthermore, because of the

highly seasonal nature of many physiological processes

in pinnipeds, the relationship of adrenal function and

photoperiod in Steller sea lions needs to be explored.Use of the reported methodology is particularly well-

suited to non-invasive acquisition of longitudinal data

necessary to investigate of the role of adrenal hormones

in Steller sea lion physiology.

Acknowledgments

All procedures were approved by Alaska SeaLife

Center and University of Alaska, Fairbanks Institu-tional Animal Care and Use Committees and were

conducted under NOAA/NMFS permits #782-1532 and

881-1668. We would like to acknowledge Carol Ste-

phens, Danielle O�Neil, Dennis Christen, Don Calkins,

Elizabeth Moundalexis, Howard Ferren, Janelle Schuh,

Jen Dailer, Jo-Ann Mellish, John Maniscalco, Lisa

Hartman, Lisa Petrauskas, Lynn Turcotte, Mary Bozza,

Matt Myers, Millie Gray, Pam Parker, Pam Tuomi,Peter Nilsson, Russ Andrews, and Tim Lebling of the

Alaska SeaLife Center, and Daniel Zatz of SeeMore

Wildlife (Homer, Alaska). Support for this project was

provided by National Oceanic and Atmospheric Ad-

ministration appropriations through the Alaska SeaLife

Center.

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