Baleen hormones: a novel tool for retrospective assessment of stress and reproduction in bowhead whales (Balaena mysticetus)
Conservation Physiology • Volume
Baleen hormones: a novel tool for retrospective assessment of stress and reproduction in bowhead whales (Balaena mysticetus)
Kathleen E. Hunt 2
Raphaela Stimmelmayr 0 1
Craig George 1
Cyd Hanns 1
Robert Suydam 1
Harry Brower Jr 1
Rosalind M. Rolland 2
0 Institute of Arctic Biology, University of Alaska Fairbanks , 902 N. Koyukuk Drive, PO Box 757000, Fairbanks, AK 99775-7000 , USA
1 Department of Wildlife Management , North Slope Borough, PO Box 69, Barrow, AK 99723 , USA
2 John H. Prescott Marine Laboratory, Research Department , New England Aquarium, Boston, MA 02110 , USA
Arctic marine mammals are facing increasing levels of many anthropogenic stressors. Novel tools are needed for assessment of stress physiology and potential impacts of these stressors on health, reproduction and survival. We have investigated baleen as a possible novel tissue type for retrospective assessment of stress and reproductive hormones. We found that pulverized baleen powder from bowhead whales (Balaena mysticetus) contained immunoreactive cortisol and progesterone that were detectable with commercially available enzyme immunoassay kits. Both assays passed parallelism and accuracy validations using baleen extracts. We analysed cortisol and progesterone at the base of the baleen plate (most recently grown baleen) from 16 bowhead whales of both sexes. For a subset of 11 whales, we also analysed older baleen from 10, 20 and 30 cm distal to the base of the baleen plate. Immunoreactive cortisol and progesterone were detectable in all baleen samples tested. In base samples, females had significantly higher concentrations of cortisol and progesterone compared with males. Cortisol concentrations in older baleen (10, 20 and 30 cm locations) were significantly lower than at the base and did not exhibit correlations with age-class or sex. Progesterone concentrations were significantly higher in females than in males at all baleen locations tested and were significantly higher in pregnant females than in non-pregnant females. Four of five mature females showed dramatic variation in progesterone concentrations at different locations along the baleen plate that may be indicative of previous pregnancies or luteal phases. In contrast, all males and all immature females had uniformly low progesterone. Baleen hormone analysis is a novel approach that, with further methodological development, may be useful for determining individual longitudinal profiles of reproductive cycles and stress responses.
Baleen; cortisol; progesterone; reproduction; stress; whales
Arctic marine mammals are faced with increasing exposure
to a variety of ecological and anthropogenic stressors, par
ticularly climate change and associated increases in seismic
exploration, oil and gas development, ship traffic and fishing
(Doney et al., 2011; Pompa et al., 2011; Reeves et al.,
2011; Moore et al., 2012)
. The potential impacts of these
rapid environmental changes on stress physiology, repro
ductive physiology and related population level effects are
largely unknown. Moreover, such physiological responses are
particularly challenging to assess in the case of mysticete
whales (baleen whales), which are difficult to study with
standard physiological techniques (Hunt et al., 2013).
Assessment of steroid hormones in novel tissue types offers
a potential solution. Conservation physiologists are increas
ingly exploring different tissue types, as diverse as urine, fae
ces, hair, blubber, respiratory vapour and earwax, to measure
steroid hormones relevant for addressing questions of stress
physiology (e.g. adrenal steroids) and reproductive physiol
(e.g. oestrogens, progestins, androgens; Amaral, 2010;
Hunt et al., 2013; Kellar et al., 2013; Trumble et al., 2013)
whales, the progestin content of faeces and blubber has been
used to diagnose pregnancy (Rolland et al., 2005; Kellar et al.,
2006, 2013), and elevated faecal glucocorticoids have been
shown to reflect exposure to various acute and chronic stress
ors (Rolland et al., 2012; Hunt et al., 2013).
Mysticete whales have a unique tissue type that has not pre
viously been explored for steroid hormone analysis, i.e. baleen.
Baleen is a stratified, cornified epithelial tissue consisting of
long, overlapping, fringed plates that grow downward from the
(Haldiman and Tarpley, 1993; Bragulla and
Homerger, 2009; Young, 2012)
, which collectively form the
whale’s filter feeding apparatus (Haldiman and Tarpley, 1993;
Werth, 2013). It has recently become clear that most cornified
epidermal tissues in vertebrates contain detectable steroid hor
mones; this has been well demonstrated in human hair and fin
gernails, mammalian fur, bird feathers and shed snakeskin
(Bortolotti et al., 2008; Warnock et al., 2010; Ashley et al.,
2011; D’Anna Hernandez et al., 2011; Lattin et al., 2011;
Bechshøft et al., 2012; Berkvens, 2013)
. The steroid content of
these cornified tissues appears to reflect circulating hormone
levels accumulated during tissue growth, e.g. human hair corti
sol content reflects adrenal activity of the previous few months,
whereas feather corticosterone content reflects adrenal activity
that occurred during feather molt
(Warnock et al., 2010; Lattin
et al., 2011; Sharpley et al., 2011)
. The glucocorticoid content
of these tissues correlates significantly with exposure to known
(Yamada et al., 2007; Van Uum et al., 2008; Fairbanks
et al., 2011; Skoluda et al., 2012)
and also with consequent
physiological impacts, such as reduced growth and body condi
tion (Lattin et al., 2011; Macbeth et al., 2012; Kennedy et al.,
2013), reduced investment in offspring (Fairhurst et al., 2012),
increased disease susceptibility (Pereg et al., 2011) and mortal
ity risk (Koren et al., 2012). Most research to date has focused
on the adrenal glucocorticoids, but preliminary results indicate
that reproductive steroids are also measurable in cornified tis
(Koren et al., 2012; Snoj et al., 2012; Caslini, 2013)
Baleen steroid analysis may offer two unusual benefits as a
conservation physiology tool. First, baleen grows continuously
from the whale’s upper palate; newer baleen is added gradually
at the base, and the oldest baleen is steadily worn off at the
(Best and Schell, 1996; Lubetkin et al., 2008; Young,
. In mature bowhead whales (Balaena mysticetus), for
example, ~15–20 cm of baleen is added per year, and a single
plate may be 4 m long; thus, one baleen plate from a mature
bowhead may represent a continuous physiological record of
approximately the past 20–25 years (Lubetkin et al., 2012; N.
Lysiak, personal communication). (Immature animals have
shorter baleen and faster baleen growth rate, and would be
expected to have maximal baleen growth records of ~10–
15 years depending on age; Lubetkin et al., 2012; N. Lysiak,
personal communication). Baleen might therefore contain
information on longitudinal profiles of past reproductive
cycles and physiological events that occurred during the last
decade or two of the whale’s life. This is of particular interest
for bowhead whales, in which reproductive cycles are still
poorly understood; for example, current estimates of calving
intervals are based on only three observed cases (Rugh et al.,
1992). A second potential advantage of baleen is that there
exist historical archives of baleen plates that date back to the
era of commercial whaling (Bockstoce, 1986), as well as mod
ern archives of baleen collected from whales that were stranded
or taken in aboriginal subsistence whaling. Steroids in corni
fied tissues often remain detectable in specimens stored long
term; museum specimens of hair and feathers that are decades
to centuries old have been found to have detectable steroid
hormones that demonstrate physiologically relevant patterns
Webb et al., 2010
Bechshøft et al., 2012
). Therefore, the pos
sibility exists that baleen hormones could be used as a valuable
tool for retrospective comparisons of steroid profiles in past vs.
present day populations, for example, comparison of inter
calving intervals (
Webb et al., 2010
Bechshøft et al., 2012
Kennedy et al., 2013).
The aim of this study was to evaluate the feasibility of
baleen as a novel sample type for assessing patterns in stress
related hormones (cortisol and its metabolites) and reproduc
tive hormones indicative of calving cycles (progesterone and
its metabolites). Our specific goals were as follows: (i) to
develop methodology for pulverizing baleen and extracting
steroids; (ii) to determine whether immunoreactive cortisol
and progesterone are detectable in baleen samples; (iii) to
validate cortisol and progesterone immunoassays for baleen
using standard parallelism and accuracy tests; (iv) to assess
patterns of baleen cortisol and progesterone at the base of the
baleen plate in relationship to current physiological status
(e.g. pregnant vs. non pregnant females); and (v) to assess
hormone variation at different locations along the baleen
plate, as a preliminary investigation to determine whether
baleen might contain a record of past physiological events.
Materials and methods
Baleen plates were collected between 2003 and 2012 from
16 subsistence harvested bowhead whales in the towns of
Barrow (n = 10), Kaktovik (n = 3), Savoonga (n = 2), and
Gambell (n = 1), AK, USA (Fig. 1). Sampling was conducted
under US National Marine Fisheries Service permit #17350
00. Whales were harvested during spring (n = 4) and autumn
months (n = 12). Baleen plates were stored at ambient tem
peratures in Barrow (annual temperature range −48 to 26°C,
with ~320 days/year below 0°C) until analysis in 2011. One
baleen plate from each whale was sampled, either by cutting
a longitudinal strip of ~40 cm length from the base of the
plate (n = 3 whales) or by using a DeWalt 18 V cordless
power drill equipped with a circular hole saw to excise a disc
of baleen ~2 cm in diameter (n = 13 whales). Note that 2 cm
represents ~1.2–1.6 months of baleen growth (Lubetkin
et al., 2008). For all whales, baleen was sampled as close to
the base of the plate as possible (i.e. most recently grown
baleen), but samples could not always be taken precisely
from the base of the plate due to fraying of baleen at the
base. In some samples, a thin dried layer of fibrous,
non baleen tissue was adhered to the base of the baleen
plate; this tissue is lighter in colour than baleen and was
discarded whenever noted, but it is possible that some sam
ples contain a small contribution from non baleen tissue.
For a subset of whales (n = 11), additional discs were
excised with the drill centred at 10, 20 and 30 cm distal to the
base (Fig. 2). This incremental sampling along the baleen plate
was carried out longitudinally (i.e. along the growth axis) as a
preliminary investigation into temporal variation in hormone
content. These distances (base to 30 cm) represent ~1.6–
2.0 years of baleen growth for most animals in our study (ani
mals >10 m body length; see Lubetkin et al., 2012) and were
selected to capture a potential endocrine record of current
pregnancies as well as pregnancies of the year before. Given
the known variation in baleen growth rate in bowheads, and
given the 2 cm diameter of our samples, the four baleen sam
pling locations are most likely to correlate to time of growth as
follows: the ‘base’ sample represents baleen grown between
0.0 and 1.2 months before death; the ‘10 cm’ location repre
sents baleen grown between 6.6 and 7.2 months before death;
the ‘20 cm’ location, between 12.6 and 15.2 months before
death; and the ‘30 cm’ location, between 18.6 and 23.2 months
before death (ranges are based on 2 cm disc diameter and on
known baleen growth rate data for bowheads of >10 m body
length; Lubetkin et al., 2008; N. Lysiak, personal communica
tion). One animal in our study was <10 m body length, a male
of 7.8 m body length; this individual may have had a faster
baleen growth rate of up to 25 cm/year (Lubetkin et al., 2008).
The final sample size was 49 subsamples from 16 baleen
plates (Table 1). All subsamples (i.e. strips and discs) were
shipped to our laboratory at the New England Aquarium
(Boston, MA, USA) for analysis.
Sex and maturity
Baleen plates were from six males and 10 females (Table 1).
Four of the females were pregnant at the time of harvest.
Sexual maturity and reproductive state were defined on the
basis of body length and relevant anatomical findings at har
vest (i.e. testicle size, ovarian morphology), as follows: imma
ture males <13.5 m body length; mature males >13.5 m;
immature females <13.5 m; mature females >13.5 m or large
ovarian follicles present; and pregnant females were identified
by presence of a fetus and/or placenta at harvest, or (if uterus
not opened) large corpus luteum and enlarged uterus (Koski
et al., 1993; George et al., 1999; O’Hara et al., 2002). Final
sample sizes were five immature males (the largest three of
which were likely pubertal; O’Hara et al., 2002), one mature
male, three immature females, three mature non pregnant
females and four pregnant females (Table 1).
Baleen sample preparation and steroid extraction
Sample preparation and extraction methods were initially
tested on a single baleen plate from a North Atlantic right
whale (NARW, Eubalaena glacialis). Isopropanol rinses
Davenport et al., 2006; Ashley et al., 2011; Bechshøft et al.,
were not used, because pilot trials showed that such
rinses had no effect on apparent cortisol content of NARW
baleen (see Supplementary Information).
We tested the following nine different methods of pulveriza
tion on NARW baleen: (i) hand mincing with scissors
ing Davenport et al., 2006)
; (ii) mortar and pestle; (iii) mortar
and pestle with liquid nitrogen; (iv) hand mincing followed by
mortar and pestle; (v) commercial blade grinder; (vi) commer
cial tobacco grinder; (vii) commercial spice grinder; (viii) labo
ratory tissue homogenizer (MP Biomedicals FastPrep 24); and
(ix) rotary electric grinder (Dremel Model 395 Type 5) with a
flexible hand held extension and tungsten carbide ball tip. The
Dremel produced the finest baleen powder with the highest
apparent cortisol concentration (see Supplementary
Information), indicating superior extraction of hormones.
Subsequently, all bowhead whale baleen samples were pulver
ized with the Dremel grinder, with the resulting baleen powder
collected on a weigh paper underneath. For the 2 cm diameter
baleen discs, several areas around the disc were pulverized to
obtain a representative sample; for the 40 cm long strips, a
2 cm diameter area at the base was pulverized.
Pilot trials on 12 bowhead samples indicated that 50 mg
samples of baleen powder sometimes had undetectably low
cortisol, whereas a 100 mg sample mass always had detect
able cortisol (see Supplementary Information). Therefore,
100 mg of well mixed baleen powder from each sample was
weighed to the nearest 0.0001 g, poured into a 16 mm ×
100 mm borosilicate glass tube, capped, and stored at room
temperature until extraction within 2 weeks.
Steroid extraction from baleen
Extraction of steroids from baleen powder followed a consen
sus protocol derived from the hair and feather glucocorticoid
(e.g. Davenport et al., 2006; Bortolotti et al., 2008)
Briefly, 4.0 ml of 100% methanol was added to 100 mg of
baleen powder, vortexed for 20 h at room temperature and
centrifuged for 15 min at 4000g. The supernatant was pipetted
into a separate borosilicate glass tube for drying, and the pellet
was rinsed twice more to recover additional hormone. For
each rinse, 1.0 ml of 100% methanol was added to the pellet,
vortexed for 30 s, centrifuged for 15 min at 4000g, and the
supernatant was transferred to the drying tube. The combined
supernatant was dried under air blow in a ThermoScientific
Abbreviations: F, female; M, male. The lengths 0, 10, 20 and 0 cm are distances from the base of the baleen plate; 0 cm is the most recently grown baleen and 30 cm
the oldest baleen tested.
a11B3 categorized as pregnant based on presence of large corpus luteum and enlarged uterus; however, abdomen was not opened at time of harvest, abdominal
organs were frozen the next day, and presence of a fetus could not be confirmed.
Reacti Therm III set at 45°C. When the volume reached
<0.5 ml, another 1.0 ml methanol was added to rinse down the
walls of the tube, and the sample was again dried. Once fully
dry, samples were reconstituted in 0.5 ml of assay buffer from
the progesterone assay kit, transferred to a cryovial and stored
at −20°C until assay within 1 week of extraction.
We tested parallelism and accuracy of bowhead whale baleen
extracts using a cortisol enzyme immunoassay (EIA; catalogue
#K003 H1) and a progesterone EIA (catalogue #K025 H1),
both from Arbor Assays (Ann Arbor, MI, USA). These two
assays were selected based on previous successful use with faeces
and respiratory vapour from other baleen whales. Cortisol
rather than corticosterone was tested based on evidence that
cortisol is probably the major circulating glucocorticoid of
(R. Rolland, unpublished data; Birukawa et al.,
. The manufacturer’s reported assay specifications are as
follows: for the cortisol assay, sensitivity = 17.3 pg/ml, limit of
detection = 45.4 pg/ml, intra assay precision = 8.8%, inter assay
precision = 8.1%; cross reactivities, dexamethasone = 18.8%,
prednisolone = 7.8%, corticosterone = 1.2%, cortisone = 1.2%
and all other tested steroids <0.1%; and for the progesterone
assay, sensitivity = 47.9 pg/ml, limit of detection = 52.9 pg/ml,
intra assay precision = 3.2%, intra assay precision = 5.7%;
cross reactivities, 3β OH progesterone = 172%, 3α OH pro
gesterone = 188%, 11β OH progesterone = 2.7%, 11α OH
progesterone = 147%, 5α dihydroprogesterone = 7.0%,
pregnenolone = 5.9% and all other tested steroids <0.1%. This
progesterone assay uses the antibody previously reported in
faecal hormone literature as ‘CL#425’ (e.g. Rolland et al.,
2005). For both assays, the manufacturer’s protocol was used
except that standards for the cortisol assay were made with the
progesterone assay buffer, based on technical advice from the
Progesterone assay parallelism was tested using three sep
arate baleen extract pools from pregnant females, non
pregnant females and males. These three pools were serially
diluted in assay buffer and assayed alongside known dose
progesterone standards. Due to low cortisol content in most
baleen samples, cortisol parallelism was tested using a single
pool from samples containing the highest cortisol content in
a pilot assay. This ‘high cortisol’ pool was serially diluted in
buffer and assayed alongside the cortisol standard curve.
Results were plotted as the percentage bound vs. the loga
rithm of relative dose, and the slopes were compared for par
allelism of the linear portion of the curve. Based on parallelism
results, all subsequent samples were assayed at 1:4 for pro
gesterone and at 1:1 (full strength extract) for cortisol; these
dilutions were chosen to fall near the 50% bound on the
standard curve, the area of greatest assay precision.
Assay accuracy was assessed by spiking standard curves
with an equal volume of a ‘low progesterone’ pool (samples
that had low progesterone in a pilot assay) diluted to 1:4, or
a ‘low cortisol’ pool (samples that had low cortisol in a pilot
assay) at 1:1, and assaying alongside standards spiked only
with assay buffer. Results were plotted as the observed dose
vs. known standard dose and assessed for linearity, slope and
All assays were performed in duplicate, with a full stan
dard curve in each assay, non specific binding wells in qua
druplicate and ‘zero’ (blank) wells in quadruplicate. Any
samples with >10% coefficient of variation between dupli
cates, or with the percentage bound <10 or >90%, were redi
luted accordingly and re assayed. Multiple samples from
the same whale (e.g. base, 10, 20 and 30 cm samples) were
assayed simultaneously in the same EIA plate, except for an
initial set of 12 base samples that were assayed on a separate
Data were analysed using InStat 3.0b for Macintosh OSX
(GraphPad Software Inc., San Diego, CA, USA). Assay paral
lelism was assessed with F tests on data for the linear portion
of the curves. Assay accuracy was assessed as follows: (i) good
ness of fit of linear regression line on expected vs. observed
dose (e.g. r2 should ideally be >0.90); (ii) slope within 0.7–1.3;
and (iii) good match of y intercept to the apparent dose when
the pool was assayed alone. Cortisol data were normally
distributed and therefore analysed with parametric tests
. Progesterone data were highly skewed and could not
be normalized with common transformations, and were anal
ysed with non parametric tests
between males vs. females and non pregnant vs. pregnant
females were analysed with Student’s t tests or Mann–Whitney
U tests. Patterns in hormones at different locations along a
baleen plate (base, 0, 20 and 30 cm) were analysed with
repeated measures ANOVA (with Tukey–Kramer post hoc
tests) or Kruskal–Wallis tests. Data are presented as
means ± SEM for normally distributed data or medians for
non normal data. The significance level was set at α = 0.05.
Both assays demonstrated good parallelism and accuracy for
bowhead baleen extracts. In the parallelism tests, the slope of
the serially diluted baleen pool(s) was not significantly differ
ent from the slope of the standard curve (for progesterone,
male pool, F1,6 = 0.0359, P = 0.856; non pregnant female
pool, F1,6 = 0.2030, P = 0.6681; and pregnant female pool,
F1,5 = 1.756, P = 0.2424; and for cortisol, F1,6 = 1.0022,
P = 0.3554; Fig. 3). In the accuracy tests, the slope of observed
vs. expected concentration was straight and was within 0.7–
1.3 for both assays, and there was a close fit between the
y intercept and the observed dose of the pool alone (proges
terone, r2 = 0.9989, slope = 0.7194, y intercept = 376 pg/ml
and pool = 424 pg/ml; and cortisol, r2 = 0.9998, slope = 1.013,
y intercept = 179 pg/ml and pool = 205 pg/ml; Fig. 3).
Cortisol (or immunoreactive cortisol metabolites) was detect
able in all baleen samples (Table 1). In base samples, females
had significantly higher cortisol than males (females,
mean = 1.72 ± 0.14 ng/g; males, mean = 0.93 ± 0.11 ng/g;
t14 = 3.819, P = 0.0019; Fig. 4). This difference disappeared
if pregnant females were excluded (mature males vs. mature
nonpregnant females, t5 = 1.777, P = 0.1358). Pregnant
females tended to have higher cortisol than non pregnant
females (Table 1), although this difference was not significant
(t8 = 1.1263, P = 0.2927), but note that statistical power for
this test was low.
Cortisol content was significantly higher in the base sam
ples than in three distal samples (older baleen at 10, 20 and
30 cm), but the distal samples from the same animals were
not significantly different from each other (within individual;
F3,10 = 12.166, P < 0.0001; P < 0.05 for post hoc comparisons
of base vs. any other location; Table 1). Furthermore, in con
trast to the sex difference seen in the base samples, males and
females did not exhibit significant differences in cortisol at
the 10, 20 or 30 cm locations.
Progesterone (or immunoreactive progesterone metabolites)
was detectable in all baleen samples (Table 1). Similar to
cortisol, females had significantly higher base progesterone
than males, and this difference disappeared if pregnant
females were excluded (females, median = 14.65 ng/g; males,
median = 6.33 ng/g; U = 9.000, P = 0.023; pregnant females
excluded and mature animals only, t5 = 0.6908, P = 0.5204;
Fig. 5). All pregnant females had higher progesterone in the
base sample than all non pregnant females, and this difference
was significant (U = 24.000, P = 0.0095).
In the baleen plates that were subsampled at multiple loca
tions, all four males and both immature females exhibited
consistently low progesterone (<20 ng/g) at all locations
(Table 1 and Fig. 5). These six animals had slightly, but sig
nificantly, higher progesterone at the base location compared
with the other three locations (F3,5 = 11.007, P = 0.0004;
Fig. 5). In contrast, four of five mature females showed
extreme variation in progesterone, spanning one to three
orders of magnitude within a single individual, at different
locations along the baleen plate (Table 1 and Fig. 5). Mature
females exhibited no relationship of progesterone with baleen
location (Fr5,4 = 2.040, P = 0.6522), and progesterone was
occasionally very high even in the oldest baleen tested (Table 1).
In the two pregnant females that were tested at multiple
baleen locations (whales 10S1 and 11B3), both had extremely
high progesterone at the base and 10 cm locations, and the
smaller whale (10S1) also had very high progesterone at the
20 cm location.
Our results indicate that immunoreactive cortisol and pro
gesterone are measurable in bowhead whale baleen using
commercially available immunoassay kits. Both hormones
appear to reflect physiological parameters of interest, such as
sex and reproductive state, with baleen progesterone exhibit
ing variation consistent with occurrence of past pregnancies.
To our knowledge, this is the first demonstration of successful
measurement of steroid hormones in baleen from any species
The higher baleen cortisol levels found in females compared
with males, at the base of the plate, is reminiscent of sex differ
ences in circulating glucocorticoids seen in many mammals
(Tilbrook et al., 2000; Panagiotakopoulos and Neigh, 2014).
Sex differences in cortisol have been documented in other cor
nified tissues as well
(e.g. polar bear hair, Bechshøft et al., 2011;
Macbeth et al., 2012; human hair, Dettenborn et al., 2012)
the present study, the highest baleen cortisol levels detected
were in pregnant females. Female mammals commonly have
elevated glucocorticoids during pregnancy
(Foley et al., 2001;
D’Anna Hernandez et al., 2011)
, and a similar pattern has been
documented in faecal glucocorticoids of North Atlantic right
whales (Hunt et al., 2006). Although preliminary, our results
indicate that baleen, at least at the base, may preserve a hor
monal signature of adrenal activity in bowhead whales.
Cortisol concentrations were markedly lower in samples
taken from older baleen (10, 20 and 30 cm) compared with
base samples. This pattern was also noted in progesterone
data, but to a lesser degree. Cortisol is one of the most polar
steroids, whereas progesterone is relatively non polar; it may
be that cortisol is more likely to leach from baleen into sea
water. Human hair loses some cortisol upon immersion in
hot water (Li, 2012), and it is unknown whether a similar
process might occur in baleen immersed in cold seawater
over months. However, any such leaching does not seem to
continue beyond 10 cm, because the 10, 20 and 30 cm sam
ples had similar cortisol concentrations. If loss of hormone to
seawater does occur, baleen from the core of the plate may be
protected from any such loss. Our samples consisted of a mix
of surface and interior baleen; future research could compare
surface vs. core baleen separately. Additionally, assay of other
adrenal steroids that are less polar than cortisol, such as cor
ticosterone and aldosterone, may prove fruitful for distin
guishing periods of higher adrenal activity in older baleen.
The dramatic variation in baleen progesterone profiles of the
mature females suggests that baleen may contain an endo
crine record of pregnancy. The progesterone profiles seen in
individual females are consistent with estimates of bowhead
gestation length and baleen growth rate (Koski et al., 1993;
Lubetkin et al., 2008). Baleen growth rate in bowheads slows
with age; for the animals sampled in this study, baleen growth
rate was estimated at ~19–20 cm/year for the smaller, younger
females and ~17–18 cm/year for the largest and oldest female
(Lubetkin et al., 2008, 2012; C. George, personal observa
tion; N. Lysiak, personal communication). For example, in
the smaller pregnant female, 10S1, the 20 cm sample was
probably grown ~12–13 months prior to death, while in the
larger pregnant female, 11B3, the 20 cm sample was probably
grown ~13.5–15 months prior to death. Given an estimated
gestation length of 13–14 months (Koski et al., 1993), it
therefore makes sense that the smaller female had high
progesterone that spanned three locations (base, 10 and
20 cm), i.e. her 20 cm sample was probably grown during
pregnancy. In contrast, the larger female had high progester
one that spanned only two locations (base and 10 cm, but
not 20 cm); thus, her 20 cm sample may represent pre preg
nancy. Although these patterns are preliminary, they suggest
that baleen may retain an endocrine signature of prior preg
nancies and that baleen should be investigated further as a
potential retrospective record of past reproductive cycles.
We note, however, that there was one pregnant female
(07B12; Table 1) that had much lower base progesterone than
the other pregnant females (though still higher than all non
pregnant females; Table 1). This female was in early preg
nancy, as determined by small fetal size (C. George, personal
observation). Lower progesterone levels might be characteris
tic of early pregnancy. Alternatively, it is possible that the base
sample of this female may not have been taken precisely from
the growth zone, due to fraying at the base of the plate (see
Materials and methods) and may represent pre pregnancy.
Overall, these data indicate that baleen progesterone
should be investigated further as a retrospective record of
prior reproductive cycles in bowhead whales. Baleen hor
mone data could be coupled with ovarian analysis (e.g. cor
pora albicans counts; George et al., 2011) and may provide
an alternative, and much needed, method of estimating inter
calving intervals and reproductive cycles in baleen whales.
Sampling at more locations along the baleen plate, ideally
coupled with stable isotope analysis to discern annual cycles
(Lubetkin et al., 2008), could help match adrenal and repro
ductive hormone profiles to season and year.
Future methodological research
The processing and extraction methods presented here pro
vide an introduction to the technique, but should not be
regarded as a final protocol. Future experiments should
include tests of other extraction methods (e.g. methanol vs.
ethanol, vortex duration, extraction temperature), with the
goal of maximizing extraction of steroids from baleen pow
der. High performance liquid chromatography or related
techniques could be used to confirm the chemical identity of
steroids present in baleen, as it is possible that immunoreac
tive steroids detected by EIA antibodies are not necessarily
pure parent hormones; unusual tissue types sometimes con
tain uncommon steroid metabolites and/or conjugated
(Wasser et al., 1988)
. Furthermore, additional anti
bodies and other quantification methods should be tested.
Other steroids could be tested as well, particularly testoster
one, estradiol, aldosterone and corticosterone.
Variation in hormone content in different sections of the
baleen plate should also be investigated, e.g. lingual vs. labial
edge, dorsal surface vs. ventral surface and surface vs. core of
plate. Potential contributions from any non baleen dried
tissue that adheres to the sample should be evaluated, as
should variation between plates from different locations in
the mouth (left vs. right side, anterior vs. posterior plates).
captive caribou and reindeer following adrenocorticotropic
hormone challenge. Gen Comp Endocrinol 172: 382–391.
In conclusion, baleen steroid analysis represents a new tool that
shows great potential for retrospective assessment of patterns of
physiological stress and reproductive cycles of baleen whales. Of
particular interest is the availability of historical baleen samples
in museum archives (samples collected during the era of com
mercial whaling) that could be used for comparisons with pres
ent day population data. Additionally, if this method proves
reliable, continued collection of baleen from modern popula
tions may allow population changes to be tracked through time
(e.g. potential shifts in average inter calving intervals), as well as
assessment of potential impacts of ongoing climate change and
increasing human activity in the Arctic. In sum, hormone analy
sis of baleen could provide an innovative means to evaluate
long term trends of stress and reproduction in whale popula
tions exposed to a changing marine environment.
Supplementary material is available
We are grateful for the co operation of the many whaling cap
tains and crews (Barrow, Kaktovik, Savoonga and Gambell)
and the Alaska Eskimo Whaling Commission for their support
and assistance in sampling landed whales. We thank the many
North Slope Borough (NSB) DWM staff and Gay Sheffield
(UAF MAP) for assistance with sample and data collection.
The NSB DWM Director Taqulik Hepa, NSB Mayor Charlotte
E. Brower, and the Steering Committee of the NSB/Shell
Baseline Studies Program provided support for this study.
Michael Macrander of Shell provided important support,
comments and guidance. We are also grateful to Jodie Treloar
(New England Aquarium) for laboratory assistance, Elizabeth
Burgess and Nadine Lysiak (New England Aquarium) for
valuable input on the manuscript and Brooke Wikgren (New
England Aquarium) for production of Figure 1.
This work was supported by the NSB [contract #2012 165 to
the New England Aquarium] with funding from the NSB/Shell
Baseline Studies Program.
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