![Regression of log-transformed mammalian body mass versus gestation period [60]. Symbols indicate the following: closed circles, terrestrial mammals; open circles, marine mammals; open triangles, other mysticete species; coloured triangles, bowhead whales (black, previous gestation estimate of 13.9 months [58]; blue, gestation estimated under hypothesis 1 (H1, this study); grey, gestation estimated under hypothesis 2 (H2, this study); red, second phase of bimodal P4 peak (this study)).](https://www.researchgate.net/publication/372648452/figure/fig5/AS:11431281177171673@1690390819971/Regression-of-log-transformed-mammalian-body-mass-versus-gestation-period-60-Symbols_Q320.jpg)
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Research
Cite this article: Lysiak NSJ, Ferguson SH,
Hornby CA, Heide-Jørgensen MP, Matthews CJD.
2023 Prolonged baleen hormone cycles suggest
atypical reproductive endocrinology of female
bowhead whales. R. Soc. Open Sci. 10: 230365.
https://doi.org/10.1098/rsos.230365
Received: 29 March 2023
Accepted: 5 July 2023
Subject Category:
Ecology, conservation, and global change biology
Subject Areas:
ecology/physiology
Keywords:
baleen, bowhead whale, calving interval,
endocrine, gestation, progesterone
Author for correspondence:
N. S. J. Lysiak
e-mail: nadine.lysiak@gmail.com
†
These authors contributed equally to this work.
Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.c.
6742244.
Prolonged baleen hormone
cycles suggest atypical
reproductive endocrinology
of female bowhead whales
N. S. J. Lysiak
1,†
, S. H. Ferguson
2
, C. A. Hornby
2
,
M. P. Heide-Jørgensen
3
and C. J. D. Matthews
2,†
1
Biology Department, Suffolk University, Boston, 02108, MA, USA
2
Arctic Aquatic Research Division, Fisheries and Oceans Canada, 501 University Crescent,
Winnipeg, Manitoba, Canada R3T 2N6
3
Greenland Institute of Natural Resources, Nuuk, Greenland
NSJL, 0000-0002-5084-432X; SHF, 0000-0002-3794-0122;
MPH-J, 0000-0003-4846-7622; CJDM, 0000-0002-8608-3905
Serial measurements of hormone concentrations along baleen
plates allow for reconstructions of mysticete whale reproductive
histories. We assessed gestation and calving interval in
bowhead whales (Balaena mysticetus) by measuring
progesterone, oestradiol, corticosterone and nitrogen stable
isotope ratios (δ
15
N) along baleen of 10 females from the eastern
Canada-west Greenland population. Three immature females
(body size < 14.32 m) had uniformly low progesterone
concentrations across their baleen, while seven mature females
(body size ≥14.35 m) had repeated, sustained elevations of
progesterone indicative of pregnancies. The mean duration of
progesterone elevations (23.6 ± 1.50 months) was considerably
longer than the approximately 14 month gestation previously
estimated for this species. We consider several possible
explanations for this observation, including delayed
implantation or sequential ovulations prior to gestation,
strategies that would allow females to maximize their fitness in
variable Arctic conditions, as well as suggest modified criteria
defining gestation as a shorter component of the entire
progesterone peak. Calving intervals varied within and among
individuals (mean = 3.7 years; range = range 2.8–5.7 years),
providing population-specific reproductive estimates for growth
models used in bowhead whale management and conservation.
1. Introduction
Life-history traits such as age of maturation and reproductive rates
are difficult to measure in large, free-ranging animals like
© 2023 The Authors. Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited.
cetaceans [1]. Reproductive knowledge of most populations comes from gross anatomical and
physiological investigations of specimens collected from commercial or indigenous whaling [2–4].
Recent studies have used endocrine markers of various tissues to study health, stress and reproductive
rates of baleen whales ([1,5–10], reviewed in [11]). Endocrine systems regulate body processes via
negative feedback loops involving steroid hormones, which ultimately control reproductive processes
such as maturation, oestrous and ovulation, pregnancy and seasonal mating behaviour [12].
For example, the female steroid hormone progesterone is produced at high concentrations during
pregnancy and is a known proxy for gestation [13,14]. Glucocorticoids (GCs), such as cortisol and
corticosterone, are biochemical markers of the vertebrate stress response, indicating activation of the
hypothalamic-pituitary-adrenal axis in response to internal or external stressors [15,16].
The filter feeding baleen plates of mysticete whales grow continuously, and can be sampled
longitudinally to construct retrospective chemical profiles to infer movement and foraging histories (via
stable isotope analysis; e.g. [17,18]) or individual life history (via endocrine analysis; e.g. [19–21]).
Validation studies have confirmed that progesterone elevations in baleen of female north Atlantic right
whales (Eubalaena glacialis) correspond with known pregnancies [20,21]. Furthermore, baleen GCs
(cortisol and corticosterone) and thyroid hormone concentrations were elevated during both internal
(i.e. pregnancy) and external (e.g. fishing gear entanglement) stressors [20–24]. Baleen of the closely
related bowhead whale (Balaena mysticetus) can exceed 4 m in length and hold upwards of 15–20 years of
tissue growth [25], allowing for retrospective reconstructions of reproductive events over biologically
meaningful time frames not possible with point measurements of tissues like skin or blubber.
Bowheads are the only Arctic-endemic baleen whale, and have evolved extreme longevity (exceeding
200 years, [26]), delayed sexual maturity (estimated at 25.8 years; [27]), and low reproductive rates
[28,29], traits that probably maximize fitness in their Arctic environment. The eastern Canada-west
Greenland (EC-WG) bowhead population is harvested seasonally in traditional Inuit hunts, with
relatively low numbers (less than 3–5) taken annually. Although current hunts are well below levels
that would induce population decline [30], EC-WG bowheads may be vulnerable to climate change-
induced shifts in sea ice parameters that impact bottom-up [31,32] and top-down [33,34] drivers of
population growth, and exacerbate anthropogenic threats such as shipping [35–37].
Population growth models require population-specific reproductive rates that are unknown for
EC-WG bowhead whales, and surrogate parameters estimated for other populations (e.g. [38]) may
differ owing to various factors (e.g. population structure and abundance, prey availability and
carrying capacity, and levels of anthropogenic disturbance). Therefore, our study objectives were to
assess gestation and associated stress responses, calving interval, and reproductive rate through
sequential measurement of steroid hormones (progesterone, oestradiol and corticosterone) along
baleen plates of 10 female EC-WG bowhead whales. In addition to informing reproductive parameters
used in population modelling, this study provides new physiological data which, when married with
existing field and gross observations, provide valuable and, until now, elusive, insights to the
reproductive endocrinology of the bowhead whale.
2. Material and methods
2.1. Baleen specimens
Baleen was collected from female EC-WG bowhead whales (n= 10) hunted by Inuit throughout the eastern
Canadian Arctic and west Greenland from 1998–2011. In Canada, six whales were hunted during July to
September from Hudson Bay, Hudson Strait, Foxe Basin, Gulf of Boothia, and northern and eastern Baffin
Island [18], and four whales were hunted in Disko Bay, Greenland in April and May [39] (table 1; figure 1).
Entire baleen plates including the most recent growth were excised from within the gum from three
whales, while only erupted baleen cut at the gumline was collected from the other seven whales (table 1).
Baleen was collected within 24–48 h of death, and frozen at –25°C. Total body length was measured for all
whales and, when possible, eye lens and reproductive tract samples were collected during necropsy to
assess whale age and reproductive status (table 1) using procedures detailed in Heide-Jørgensen et al. [40].
2.2. Baleen sampling
Each baleen plate was cleaned using water and scrubbing pads, then scraped using a scalpel blade
to remove adhered surface material. Starting at the proximal end, baleen samples were drilled every
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
2
2 cm along the outside edge using a hand-held rotary tool fitted with a 1/16-inch drill bit. Samples were
collected until the tapered (distal) end of the baleen became too thin to collect the required 100 mg for
hormone analysis, which typically excluded the final 10–20 cm of each plate. The 2 cm sample
increments represent sub-seasonal, near monthly temporal resolution given annual baleen growth rates
of 14–25 cm yr
−1
in bowhead whales [17,18].
2.3. Hormone extraction
Baleen hormone extraction followed procedures developed by Hunt et al. [19,20,22,41], with some
modifications. Six millilitres of 100% high-performance lipid chromatography-grade methanol was
added to 100 mg of each baleen sample in 16 × 100 mm borosilicate glass tubes, which were capped
and vortexed for 2 h, and then centrifuged for 15 min at 3000g. Then, 3.75 ml of the methanol
supernatant containing hormones was transferred to a second borosilicate tube, dried down under an
N
2
stream for approximately 6 h, and frozen at –80°C. Dried hormone extracts were reconstituted in
1000 µl of enzyme immunoassay buffer (catalogue ‘X065’, Arbor Assays, Ann Arbor, MI, USA),
vortexed, and pipetted to a cryovial for storage at –80°C prior to hormone assay. This solution is
herein termed the ‘1:1’(full-strength, neat) extract.
2.4. Hormone assays
Commercial enzyme immunoassay kits from Arbor Assays were used to analyse baleen progesterone
(catalogue no. K025), corticosterone (catalogue no. K014) and 17β-oestradiol (catalogue no. K030).
Progesterone and corticosterone were measured in all samples (i.e. at 2 cm increments), while
oestradiol was measured at lower resolution (every 4 to 6 cm) that was increased to every 2 cm during
Table 1. Bowhead whale metadata, including hunt location and date, morphology, age, and gross examination of reproductive
tracts, ranked by body size per sampling country. NU, Nunavut; QC, Quebec; GL, Greenland.
whale identity hunt location date
total
length
(m)
AAR
age
(yr)
a
baleen
length
(cm)
a
corpus lutei
detected
(Y/N)
fetus
detected
(Y/N)
EASTERN CANADIAN ARCTIC
NSA-2011-03 Gulf of Boothia,
Kugaaruk, NU
08/2011 9.04 nd 176nd nd
IG-HB-2002-001 Foxe Basin, Iglooik,
NU
08/2002 14.19 32.0 263+ nd nd
NSA-2010-02 Foxe Basin, Repulse
Bay, NU
08/2010 14.32 nd 296nd nd
NSA-2009-02 Hudson Strait,
Rankin Inlet, NU
08/2009 16.15 45.1 232+ nd nd
RB-2005-001 Foxe Basin, Repulse
Bay, NU
08/2005 16.40 64.0 267+ nd nd
01-2009 Hudson Strait,
Kangiqsujuak, QC
08/2009 17.29 139.3 338nd nd
GREENLAND
500 Disko Bay, GL 04/2010 14.35 36.4 236+ N N
325 Disko Bay, GL 05/2009 15.50 41.9 192+ nd nd
501 Disko Bay, GL 04/2010 15.85 40.8 262+ Y N
502 Disko Bay, GL 05/2010 16.10 32.7 284+ Y N
a
AAR: age estimate via aspartic acid racemization of the eye lens; total length (embedded+erupted baleen); +erupted length;
nd: not determined.
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3
some periods of elevated progesterone. An extensive laboratory validation study by Hunt et al. [41]
demonstrated that all three assay antibodies exhibited reliable binding affinity to the desired hormone
in bowhead whale baleen (i.e. good parallelism), and verified that each assay was able to distinguish
a range of concentrations with acceptable accuracy.
The manufacturer’s protocols were followed for analysis of all three hormones. Samples, standards,
non-specific binding, and blank wells were assayed in duplicate. Any samples that fell outside 10–90%
bound on the standard curve were re-assayed; samples with high hormone concentrations ( per cent
bound < 10%) were diluted 2-fold (1 : 2), or up to 1 : 128 in some high progesterone samples, while
samples with low hormone concentrations ( per cent bound > 90%) were concentrated within the assay
wells at 2 : 1. Samples with more than 10% coefficient of variance between duplicates were re-assayed.
Results were converted to nanograms of immunoreactive hormone per gram (ng g
−1
) of baleen.
Baseline hormone concentrations were determined for each dataset using an iterative process
excluding all points that deviate from the mean by 2 s.d. until no points exceed this threshold (after [42]).
2.5. Stable isotope analysis
Previous stable nitrogen isotope (δ
15
N) measurements along the baleen of the six whales hunted in
Canada indicated annual cycling that most likely reflected foraging in isotopically distinct summering
and wintering areas of their annual range [18]. We used these δ
15
N cycles to estimate annual baleen
growth rates (see below) to interpret variation in hormone concentrations within a temporal context.
δ
15
N of the four additional whales hunted in Greenland were measured at the same laboratory
following the same protocols as the other six animals (details in [18]).
2.6. Data analysis
Average annual baleen growth rate across the entire baleen plate of each whale was estimated
from spectra of autoregressive (AR) models [43,44] fitted to annual δ
15
N cycles (see [18]). Period
60° N
65° N
60° W80° W
latitude
longitude
70° N
Figure 1. Bowhead whale hunt locations. Shading indicates approximate bowhead whale seasonal distribution in summer (red) or
winter (green).
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
4
(i.e. annual baleen growth rate) was estimated by converting the spectral peak frequency of each
modelled hormone profile to samples per period (1/peak frequency) and then multiplying by the
2 cm sample increment.
We tested two hypotheses for interpreting progesterone (P4) peaks, areas of elevated P4 concentration
typically indicative of pregnancy status. Under hypothesis 1, we defined gestation as regions of baleen
where P4 concentrations became elevated 2 standard deviations above baseline, remained so for more
then 2 consecutive samples, and then returned to values at or below baseline (figure 2). The first and
second phases (i.e. minima to minima) of the bimodal P4 peaks were also identified (see vertical
dashed line, figure 2). Under hypothesis 2, peaks were defined similarly, but gestation ended when the
final P4 maxima was reached—such that points on the decline back to P4 baseline values were
excluded from calculations of gestation (figure 2). Only complete P4 peaks were included in
subsequent analyses of gestation and calving interval. To estimate gestation, the length of baleen
tissue containing each P4 peak was divided by the annual baleen growth rate (δ
15
N period). Interpeak
interval was defined as a region of baleen following a P4 peak until the initiation of a subsequent
peak (figure 2). To estimate the duration of each interpeak interval, the length of baleen separating P4
peaks was divided by the annual baleen growth rate, or δ
15
N period. Calving interval, or the time
from one calving event to the next, was defined as gestation + the subsequent interpeak interval
(figure 2).
3. Results
The baleen of seven of the 10 females, ranging in length from 14.35–17.29 m, contained repeated,
sustained elevations in P4 (figures 3 and 4). In general, P4 concentration increased from less than 10
to more than 100 ng g
−1
during each elevation. Initial increases in baleen P4 tended to coincide with
an increase towards seasonal maximum δ
15
N ratios, indicative of the late winter/early spring season
(e.g. figure 2). Progesterone peaks were bimodal; with P4 being the only hormone with sustained
14
15
10
5
01
10
100
progesterone (ng g
–1
)
1000 gestation period interpeak interval
calving interval
(a)
(b)
180200 160
baleen length (cm)
13
12
corticosterone (ng g
–1
)
oestradiol (ng g
–1
)
G
15
N (‰)
G15N period
Figure 2. (a) Patterns in stable nitrogen ratios (δ
15
N, green) and (b) concentrations of immunoreactive progesterone (black),
corticosterone (red) and oestradiol (blue) across a representative section of bowhead whale baleen (from individual 500). Closed
circles indicate hormone concentrations above a baseline threshold, while open circles indicate hormone concentrations within
baseline levels. Dark grey box highlights gestation as defined under hypothesis 2; light grey box extends gestation as defined
under hypothesis 1. Light grey vertical dashed line demarcates first and second phase of P4 peak.
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
5
elevations above baseline in the first phase of the peak. Corticosterone (B) peaks were observed in the
second phase, and these persisted to the end of the P4 peak (figures 2–4). Oestradiol was frequently
elevated during the second phase of bimodal P4 peaks (figures 2–4).
Under hypothesis 1 (gestation includes all P4 values above baseline), gestation of the seven adult
female whales lasted a mean of 23.6 ± 1.5 months (range = 21.6–25.6 months; table 2). The mean
duration of the first phase of the P4 peak was 10.0 ± 1.4 months (range = 8.0–12.0 months; table 2),
and the mean duration of the second phase of the P4 peak was 13.6 ± 1.5 months (range = 11.6–16.0
months; table 2) The mean interpeak interval was 20.9 ± 11.1 months; with considerable intra- and
inter-individual variability (range 12.1–43.3 months; table 2). Under hypothesis 2 (gestation ends at
the last P4 maxima), mean gestation was 16.2 ± 1.5 months (range 14.3–19.2 months; table 2). The
mean interpeak interval was 28.1 months, again with considerable intra- and inter-individual
variability (range 19.1–49.2 months; table 2). The single shortest interpeak interval observed was 2.8
or 8.8 months (depending on how gestation was defined; individual 502) and the single longest was
48.3 or 53.3 months (4.0 or 4.4 years; individual NSA 2009-02) (figures 3 and 4). Given these
observations, under either definition of gestation, the average calving interval (i.e. gestation+
interpeak interval) for the seven whales was 3.7 years (range 2.9–5.7 years; table 2).
0
5
0
10
15
20
0
5
0
10
15
20
corticosterone (ng g
–1
)
oestradiol (ng g
–1
)
baleen length (cm)
baleen length (cm)
0100200300
1
0100200300
100200300
100200300
5
0
10
15
20
10
100
1000
1
10
100
1000
5
0
10
15
20
corticosterone (ng g
–1
)
oestradiol (ng g
–1
)
progesterone (ng g
–1
)
1
10
100
1000
1
10
10
12
14
16
10
12
14
16
10
12
14
16
10
12
14
16
500
501 502
325
(a)(b)
(c)(d)
100
1000
progesterone (ng g
–1
)G
15
N (‰)G
15
N (‰)
Figure 3. Profiles in stable nitrogen ratios (δ
15
N, green) and concentrations of immunoreactive progesterone (black), corticosterone
(red) and oestradiol (blue) across the baleen plates of female bowhead whales hunted off west Greenland. Closed circles indicate
hormone concentrations above a baseline threshold, while open circles indicate hormone concentrations within baseline levels. Dark
grey box highlights gestation as defined under hypothesis 2; light grey box extends gestation as defined under hypothesis 1.
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
6
0
5
0
10
15
20
baleen length (cm)
100200300
1
10
100
1000
progesterone (ng g
–1
)
10
12
14
16 NSA 2009 02
(d)
G
15
N (‰)
0
5
0
10
15
20
baleen length (cm)
100200300
1
10
100
1000
10
12
14
16 RB 2005 001
(e)
0
5
0
10
15
20
baleen length (cm)
100200300
1
10
100
1000
10
12
14
16 01 2009
corticosterone (ng g
–1
)
oestradiol (ng g
–1
)
0
5
0
10
15
20
100200300
1
10
100
1000
progesterone (ng g
–1
)
10
12
14
16 NSA 2011 03
(a)
G
15
N (‰)
0
5
0
10
15
20
100200300
1
10
100
1000
10
12
14
16 IG HB 2002 001
(b)
0
5
0
10
15
20
100200300
1
10
100
1000
10
12
14
16 NSA 2010 02
(c)
corticosterone (ng g
–1
)
oestradiol (ng g
–1
)
(f)
Figure 4. Profiles in stable nitrogen ratios (δ
15
N, green) and concentrations of immunoreactive progesterone (black), corticosterone
(red) and oestradiol (blue) across the baleen plates of female bowhead whales hunted in the eastern Canadian Arctic. Closed circles
indicate hormone concentrations above a baseline threshold, while open circles indicate hormone concentrations within baseline
levels. Dark grey box highlights gestation as defined under hypothesis 2; light grey box extends gestation as defined under
hypothesis 1.
Table 2. Estimates of baleen stable nitrogen isotope (δ
15
N) period and mean duration of gestation, interpeak, and calving
intervals of individual bowhead whales. (Reproductive cycle parameters were estimated under two hypotheses: (H1) inclusive of
total progesterone (P4) peak and (H2) inclusive of P4 peak up to final maxima. Immature whales were not included in this
analysis. nd, not determined.)
whale sample
identity
δ
15
N
period
(cm yr
−1
)
H1: P4 total H2: P4 max
calving
interval
(years)
full P4
peak
(months)
P4 1
st
phase
(months)
P4 2
nd
phase
(months)
interpeak
interval
(months)
gestation
(months)
interpeak
interval
(months)
NSA-2011-03 nd nd nd nd nd nd nd nd
IG-HB-2002-001 20.2 nd nd nd nd nd nd nd
NSA-2010-02 26.3 nd nd nd nd nd nd nd
NSA-2009-02 14.4 25.0 10.8 14.2 43.3 19.2 49.2 5.7
RB-2005-001 15.0 25.6 12.0 13.6 26.7 16.8 35.2 4.3
01-2009 16.0 24.0 11.0 13.0 13.9 14.3 23.3 3.1
500 15.9 21.6 10.0 11.6 20.0 15.5 26.0 3.5
325 16.0 22.0 10.0 12.0 18.0 16.0 24.0 3.3
501 17.9 22.8 8.3 14.5 12.1 15.4 19.1 2.9
502 17.4 24.0 8.0 16.0 12.4 16.0 20.0 3.0
mean 16.1 23.6 10.0 13.6 20.9 16.2 28.1 3.7
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
7
The remaining three females, ranging from 9.04–14.32 m in total length, had uniformly low P4
concentrations across the majority of their baleen plates (figure 4). One of these females (IG-HB-2002-
001, total length = 14.19 m) exhibited one bimodal P4 peak in its most recent growth (0–46 cm;
figure 4b). Although probably indicative of this whale’s first pregnancy, this P4 peak was incomplete
since its baleen was excised at the gumline and excluded unerupted baleen and was therefore
excluded from analyses of gestation events (see Methods). The other two females (NSA-2011-03 and
NSA-2010-02) had sporadic elevations in P4 and corticosterone, but neither exhibited sustained P4
peaks (figure 4).
4. Discussion
The prevailing paradigm of mammalian reproductive physiology indicates that P4 is elevated during
pregnancy [45,46]. In free-ranging large whales, high P4 (i.e. several orders of magnitude higher than
in individuals of other reproductive states) has been linked to gestation (reviewed in [11], but see
[47]). Accordingly, pregnancy in several mysticete species has been assessed via P4 analysis of skin
[48], blubber [5,49–51], faeces [52], respiratory vapour [53,54], and baleen [19–24]. Applying the
conventional interpretation that high tissue P4 indicates pregnancy status (i.e. hypothesis 1), we
would infer that bowhead whale gestation lasts 23.6 months—considerably longer than previous
estimates of 13.9 months, based on observations of mating season, fetal growth and parturition dates,
and Bayesian modelling [55–58]. The duration of progesterone peaks was determined using annual
baleen growth rates—inferred from the periods of δ
15
N cycles derived from autoregressive models
fitted across an entire baleen plate. This analysis could potentially obscure interannual variability in
baleen growth related to ageing [59] or physiological changes, including pregnancy or lactation.
However, we typically observed two annual δ
15
N cycles coincident with progesterone peaks
(figures 2–4), confirming that baleen growth rate did not increase substantially over periods of
elevated progesterone concentrations. Furthermore, bowhead whales, like other mysticetes, have
shorter gestations than expected based on their body size, and a longer gestation better fits allometric
predictions (figure 5; data from [60]). However, bowheads and other mysticetes experience seasonality
thought to constrain their reproduction.
Field observations of bowhead mating behaviour (January–May) and parturition (late spring-early
summer; [29]) could align with a gestation lasting approximately 2 years, but observed trends in fetal
growth do not. Decades of observations following subsistence hunts reveal that bowhead whale
fetuses are either small (fetal length < 100 cm) or large (greater than 350 cm) in the spring, and of
intermediate size (approx. 200–300 cm) in the autumn [39,57,58]. Neonate calves, born in the late
spring to early summer, are more than 400 cm in total length. If gestation was extended over nearly 2
years, we would expect to observe fetuses of intermediate length in the spring following the first year
log
gestation period (days)
2
456
terrestrial
y = 0.18x + 1.34
R2 = 0.72
marine
mysticete
bowhead whale [58]
H1, this study
H2, this study
P4 1st phase, this study
78
3
log
body mass (g)
Figure 5. Regression of log-transformed mammalian body mass versus gestation period [60]. Symbols indicate the following: closed
circles, terrestrial mammals; open circles, marine mammals; open triangles, other mysticete species; coloured triangles, bowhead
whales (black, previous gestation estimate of 13.9 months [58]; blue, gestation estimated under hypothesis 1 (H1, this study);
grey, gestation estimated under hypothesis 2 (H2, this study); red, second phase of bimodal P4 peak (this study)).
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
8
of gestation, as well as bimodal variation in the lengths of fetuses in the autumn (representing the first
and second autumn after conception).
Given these observations, an approximately 2 year gestation is plausible with the occurrence of a
reproductive delay (in fertilization or implantation), a period of polyestry, or both. Delayed
fertilization may provide opportunities for post-copulatory sexual selection, including sperm
competition [61,62] in polyandrous species such as balaenid whales where one female mates with
many males in quick succession. Male balaenids, including bowhead and right whales, have larger
than expected testes [63–66] and experience seasonal increases in testosterone production [67,68],
suggesting the importance of sperm competition. Delayed implantation can allow females to match
the energetic demands of pregnancy and lactation with favourable environmental conditions
and optimal food abundance, or to selectively abort an embryo in suboptimal conditions [61,69].
Delayed implantation is common in some mammalian orders (e.g. pinnipeds, mustelids, ursids;
reviewed in [61,70]), but has only been observed in one cetartiodactyl species, the roe deer (Capreolus
capreolus; [71,72]). Delayed implantation is not thought to occur in cetaceans (reviewed in [2,73]),
although gross inspections of whale carcasses could overlook small dormant blastocysts in pregnant
females. Species exhibiting delayed implantation exhibit surges in P4 and/or oestradiol with
implantation and the transition to active gestation [74,75], which is not inconsistent with findings of
this study: the second phase of bimodal P4 peaks have a P4 surge and concurrent oestradiol
elevations (figures 2–4).
An alternative explanation is that the baleen P4 peaks reflect a period of prolonged oestrus or sequential
ovulations (seasonal polyestry, see [3]) preceding gestation. Progesterone elevations can occur in advance
of implantation owing to the secretions of the corpus luteum (CL), atemporary endocrine structure formed
from the ruptured ovarian follicle following ovulation. The CL secretes P4 to prepare the uterine lining for
the implantation of a blastocyst [12], and continues to secrete P4 during early pregnancy prior to
development of the placenta. High tissue P4 values have been associated with ovulation in cetaceans
[50,53,76], with sequential ovulations and concurrent P4 elevation for 86 and 103 days, respectively, in
captive odontocetes [77]. Mature bowhead whale ovaries can contain several corpus albicantia (CAs;
formed from the involution of a CL) of similar size in the same ovary, signalling that several ovulations
occurred close in time [29,78]. Bowhead whale mating behaviour has been observed from winter
through to summer (M.P. Heide-Jørgensen 2018, unpublished data), and mating behaviour of north
Atlantic right whales has been observed year-round [79–81]. A prolonged oestrous season in these
species could allow females to maximize their reproductive success by being sexually receptive over
longer time periods and capitalizing on multiple copulations and/or sperm competition to determine
the highest quality father for their offspring.
The bimodal shape of the baleen P4 peaks, coupled with elevated corticosterone (B) and oestradiol
during the second half (figures 2–4), point to a biphasic event consistent with either interpretation
outlined above, in which the second phase of a P4 peak represents the active gestation phase.
Glucocorticoids like corticosterone play an important role in fetal organ development during gestation
[82,83]. Moreover, oestrogens (the most prominent being oestradiol) increase rapidly during the
middle of pregnancy and remain elevated until parturition [84]. The second phase of the P4 peaks
(with coincident corticosterone and sporadic oestradiol elevations) had a mean duration of 13.6
months (range = 11.6–16.0 months; table 2; figures 3 and 4), which aligns more closely to the field-
derived estimate of an approximately 14 month gestation for bowhead whales. Previous work on
north Atlantic right whales by Lysiak et al. [21] and Hunt et al. [22] similarly observed (i) baleen P4
peaks that are markedly longer (range = 15–23 months, n= 3 individuals) than gestation estimates
derived from field observations and gross anatomy (12–13 months; [80,85]), and (ii) sustained
elevations of corticosterone and oestradiol in the second phase of P4 peaks. Similar observations in
two closely related species suggest certain unique aspects of balaenid whale endocrine profiles.
Alternatively, hypothesis 2 (in which we define gestation as a shorter component of the overall P4
peak) provides a mean gestation of 16.2 ± 1.5 months (range = 14.3–19.2 months; table 2), also closer to
field-derived estimates of approximately 14 months [58]. This interpretation follows the logic that as
pregnancy progresses, the placenta becomes a principal tissue of steroidogenesis [86,87]. Following
expulsion of the placenta during parturition, circulating levels of P4 return to baseline levels [50]; but
given their massive body size, the half-life of circulating hormone levels could be substantial in
bowhead whales. Although more in alignment with current gestion estimates, this interpretation
provides no explanation for the bimodal nature of the P4 peak, nor the concurrent increases in the
other hormones in its latter half. Further work examining longitudinal endocrine profiles, analysing
additional hormones, more individuals, and additional species is needed to better understand baleen
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
9
whale reproductive physiology, including the ways in which they may diverge from the generalized
mammalian endocrine model.
Competing interpretations of baleen hormone profiles with respect to gestation do not impact
estimates of calving interval (only how it is partitioned between gestation and interpeak interval). The
mean calving interval of 3.7 years (range 2.8–5.7 years) across the seven mature individuals is in close
agreement with previous estimates for the Bering-Chukchi-Beaufort (BCB) bowhead population of 3–4
years [88], 3.06 years [89], or 3.15 years [38] determined using data from postmortem observations,
ice-based censuses, and aerial surveys. However, we observed variation within and among
individuals (table 2), which could reflect the influence of age/experience, body size, environmental
conditions, and resource availability. Like all mysticetes, bowhead whales are capital breeders that
rely on a thick blubber layer to support gestation and lactation [90–93], and previous studies have
found that variable birth intervals in bowheads may cycle with climatic oscillations [56] or other
indices of food availability.
We observed some deviations from the typical pattern of baleen P4 expression in two females and cite
them as evidence of potential reproductive dysfunction or pregnancy loss. Female 01-2009 had a single,
sustained P4 elevation for approximately 7 years (from baleen length 100–212 cm) with some coincident
corticosterone elevations and sporadic increases in oestradiol (figure 4f). This region of baleen may
indicate approximately 5 years of unsuccessful reproductive cycles or pregnancies, finally ending in an
approximately 2 year gestation phase (as defined under both hypotheses explored in this paper) from
baleen length 100–128 cm. Female 502 had the single shortest interpeak interval at 2.8 or 8.8 months
(depending on how gestation was defined, from baleen length 98–106 cm; figure 3d). The interpeak
interval includes lactation as well as a recovery period following weaning, where adult females
replenish their lipid reserves and energy capital for subsequent calving events [90–93]. If peri- or
neonatal mortality occurs, a female could conceivably be in positive energy balance and may be able
to support a new pregnancy without a prolonged recovery period.
The uniformly low P4 concentrations of three females ranging from 9.04 to 14.32 m in length indicate
these animals were immature. For comparison, examination of ovaries for CL or CA in the BCB bowhead
whale population indicated that females less than 12.6–14.2 m were sexually immature [29,78]. Of these
three females, IG-HB-2002-001 had baleen containing one P4 peak near the proximal (most recent)
growth, which we interpret as her first pregnancy. Aspartic acid racemization of the eye lens from this
animal estimated her age at 32 years and given that the unerupted baleen tissue from this animal was
not sampled, we estimate that her age conception was approximately 28–29 years (table 1; figure 4).
IG-HB-2002-001 had elevated oestradiol in the approximately 1.5 years preceding conception,
indicating that she may have entered sexual maturity. We did not quantify oestradiol in older portions
of this animal’s baleen, so an accurate estimate of age at sexual maturity is not possible in this case,
though our observations are in line with previous field studies estimates of bowhead whale sexual
maturity at approximately the age of 26 [27,78,88]. Future work directly investigating endocrine
markers of sexual maturity is needed for this species. The inferred immature status (or recently
mature whale IG-HB-2002-001) of these whales is consistent with independent studies that reveal
spatial segregation by sex, age and reproductive status among EC-WG bowhead whales. These three
whales were among four collected from Foxe Basin and Gulf of Boothia, which are known
aggregation areas for juvenile bowhead whales [94]. Moreover, upwards of 85% of animals that
aggregate at Disko Bay, Greenland, are mature females [95], which is also consistent with the regular
P4 peaks exhibited in baleen of each of the four animals sampled there.
5. Conclusion
This study reports on novel, retrospective, longitudinal baleen endocrine profiles from 10 female
bowhead whales, providing, to our knowledge, the first population-specific calving interval estimates
for EC-WG bowhead whales. The extended, bimodal P4 peaks in mature female baleen suggest that
bowhead whale reproductive cycles are longer and more complex than previously assumed. We
suggest these bimodal P4 peaks potentially reflect a biphasic event, with the first component
corresponding to either a period of delayed implantation, development, or prolonged oestrus followed
by active gestation, marked by concurrent elevation of all three hormones—progesterone,
corticosterone, and oestradiol. Evolutionarily, both strategies have advantages which would allow
females to maximize their reproductive success in Arctic environments with high interannual
variability in environmental conditions. Alternatively, longitudinal P4 records of gestation may be
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230365
10
more appropriately defined as ending prior to tissue hormone concentrations fully returning to baseline
values. Future studies should examine additional hormones that could be included in the baleen
endocrine profile (e.g. prolactin, relaxin or other progestagens), including higher temporal resolution
analysis of oestrogens, to better assess evidence (or lack thereof) for delayed implantation or polyestry
in this species. Our results warrant consideration for future endocrine studies of cetacean tissues,
especially in point samples from matrices like skin, blubber, faeces or respiratory vapour—as high
tissue P4 concentrations may not mean that a female mysticete is currently pregnant (e.g. if
hypothesis 2 is correct; see [50,53,87]). Furthermore, the observed elevations in corticosterone
coincident with gestation, an internal stressor, are important to consider in the broader context of
marine mammal stress physiology, which often connects elevated glucocorticoids to external stressors
only. Our integration of high-resolution longitudinal hormone data from baleen, along with
consideration of published field and gross observations from the BCB and EC-WG bowhead
populations, reveals novel aspects of bowhead reproductive physiology that appear to diverge from
the generalized mammalian reproductive endocrinology model.
Ethics. All baleen used in this study was collected from legally hunted bowhead whales in Nunavut and Nunavik
(Canada) and Greenland. Local Hunters and Trappers Organizations (HTOs) and wildlife officers in their
respective communities collected specimens in collaboration with Fisheries and Oceans Canada and the Greenland
Institute of Natural Resources. Baleen collected in Greenland was exported to Canada under CITES Certificate of
Scientific Exchange (COSE) permits DK003 and CA025.
Data accessibility. Data have been provided in this submission as a table/electronic supplementary materials [96].
Authors’contributions. N.S.J.L.: conceptualization, formal analysis, investigation, methodology, validation, visualization,
writing—original draft, writing—review and editing; S.H.F.: conceptualization, funding acquisition, resources,
writing—review and editing; C.A.H.: investigation, writing—review and editing; M.P.H.-J.: funding acquisition,
resources, writing—review and editing; C.J.D.M.: conceptualization, funding acquisition, investigation,
methodology, resources, writing—original draft, writing—review and editing.
All authors gave final approval for publication and agreed to be held accountable for the work performed therein.
Conflict of interest declaration. We declare we have no competing interests.
Funding. This research was funded by Fisheries and Oceans Canada (DFO) to S.H.F. and C.J.D.M., and N.S.J.L. was
supported by a fellowship from Suffolk University.
Acknowledgements. We thank the following organizations and individuals in Canada and Greenland for collecting and
making baleen available for scientific study: Hunters and Trappers Organizations (HTOs); wildlife officers in their
respective communities across Nunavut and Nunavik; Fisheries and Oceans Canada (DFO) offices in Iqaluit, NU,
Mont-Joli, QC (P. Carter) and Winnipeg, MB (L. Dueck, B. Dunn, P. Hall, T. Kelley and O. Nielsen), and Makivik
Corporation in Kuujjuaq, QC (M. Simard); N. Levermann of the Government of Greenland and all whaling crew
and community members. T. Naaykens and C. Willing assisted with baleen sampling and/or hormone extraction.
K. Hunt (George Mason University) provided invaluable advice on hormone extraction methods. K. Campbell
(University of Massachusetts Boston) provided laboratory space and equipment for hormone analysis. Stable isotope
analysis was conducted by P. Middlestead, W. Abdi and P. Wickham at the University of Ottawa. Finally, we thank
three anonymous reviewers who improved this manuscript with their thoughtful suggestions.
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