Use of hormones in assessing reproductive physiology of humpback whales (Megaptera novaeangliae) from Juneau, Alaska

Abstract

Humpback whales (Megaptera novaeangliae) in Southeast Alaska have been studied for over 50 years, and are largely considered a recovery success since the cessation of commercial whaling. Reproductive physiology is an important factor to consider in studying population health and can provide important insights into the drivers contributing to population abundance fluctuations. Validated assays for progesterone and testosterone were used on blubber biopsies from humpback whales (N = 33 whales, 71 samples) near Juneau, Alaska, in 2020 and 2021. Long-term sighting histories were used to confirm detected pregnancies with calf sightings the following year. Blubber samples were divided into two seasonal bins (early and late summer). Pregnant females sampled in both early and late summer of both 2020 and 2021 showed elevated progesterone concentrations compared to other reproductive states (p

Full text

Theriogenology Wild 3 (2023) 100050
Available online 14 August 2023
2773-093X/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Use of hormones in assessing reproductive physiology of humpback whales
(Megaptera novaeangliae) from Juneau, Alaska
S. Atkinson
a
,
*
, V. Melica
a
, S. Teerlink
b
, K. Mashburn
a
, J. Moran
c
, H. Pearson
d
a
University of Alaska Fairbanks, College of Fisheries and Ocean Sciences, 17101 Point Lena Loop Road, Juneau, AK 99801, USA
b
National Marine Fisheries Service, Alaska Regional Ofce, 709 West 9th Street, Juneau, AK 99801, USA
c
Alaska Fisheries Science Center, National Oceanic and Atmospheric Administration, Juneau, 17109 Pt. Lena Loop Road, Juneau, AK 99801, USA
d
University of Alaska Southeast, Department of Natural Sciences, 11066 Auke Lake Way, Juneau, AK 99801, USA
ARTICLE INFO
Key words:
Humpback whale
Megaptera novaeangliae
Progesterone
Testosterone
Pregnancy rate
Calving rate
Reproduction
Cetacean
Blubber biopsy
ABSTRACT
Humpback whales (Megaptera novaeangliae) in Southeast Alaska have been studied for over 50 years, and are
largely considered a recovery success since the cessation of commercial whaling. Reproductive physiology is an
important factor to consider in studying population health and can provide important insights into the drivers
contributing to population abundance uctuations. Validated assays for progesterone and testosterone were used
on blubber biopsies from humpback whales (N =33 whales, 71 samples) near Juneau, Alaska, in 2020 and 2021.
Long-term sighting histories were used to conrm detected pregnancies with calf sightings the following year.
Blubber samples were divided into two seasonal bins (early and late summer). Pregnant females sampled in both
early and late summer of both 2020 and 2021 showed elevated progesterone concentrations compared to other
reproductive states (p <0.05). Progesterone concentrations in adult male whales (0.3 ±0.2 ng/g) were not
signicantly different from lactating or resting female whales. Blubber testosterone concentrations in adult male
humpback whales ranged from 0.05 to 1.1 ng/g, and mean concentrations were approximately double those of
female whales in any reproductive state. Pregnancy was detected in 5 of 11 and 4 of 9 adult females in 2020 and
2021 respectively, yielding summer season pregnancy rates for sexually mature females at 0.45, and 0.44,
respectively. Calving rates were 0.36 and 0.22 in 2020 and 2021, respectively, and the annual growth rate for
this subpopulation was calculated at 2.6 % per annum. One female had successful pregnancies for four
consecutive years. These results demonstrate the synergistic value of combining immunoreactive assays and
long-term sighting histories to further knowledge of reproductive physiology in individual humpback whales,
which can be expanded to assessing the health of a population or ecosystem.
1. Introduction
Steroid hormones have been measured in a variety of tissues in
multiple marine mammal species, including baleen whales [1–16].
When available, blood serum, blubber, and feces are the matrices of
choice for investigating endocrine response to relatively recent events
(minutes to days) in living marine mammals [10–12,17–25]. Especially
for large whales, many studies have focused on blubber, as samples can
be obtained with minimally invasive techniques and reect
close-to-recent physiological processes [4–7,26–29]. Blubber is common
to most marine mammals, and it consists of a vascularized layer of fat
and connective tissue beneath the skin [30]. Energy storage is a major
function for this tissue; however, it also serves as a thermal insulator and
aids in buoyancy and hydrodynamics. Being a dynamic tissue, blubber
changes in response to reproductive status, nutritional status, and age
[30]. Steroid hormones are synthesized from cholesterol and because of
their lipophilic nature, blubber is a suitable tissue for endocrine analysis.
Additionally, studies have shown that accumulation of steroids in this
tissue occurs within hours from their secretion in blood [19,26] sug-
gesting that blubber can be used to describe recent physiological events.
Progesterone is a steroid hormone secreted by the corpora lutea in
the ovaries during the estrous cycle, and is the predominant compound
responsible for sustaining pregnancy in whales and many other mam-
mals [7,31,32]. In general, progesterone concentrations are low in
sexually immature whales and elevated in sexually mature females [1,2,
4,7,25]. Progesterone concentrations in blubber have been applied as a
* Corresponding author.
E-mail address: skatkinson@alaska.edu (S. Atkinson).
Contents lists available at ScienceDirect
Theriogenology Wild
journal homepage: www.journals.elsevier.com/theriogenology-wild
https://doi.org/10.1016/j.therwi.2023.100050
Received 6 March 2023; Received in revised form 1 August 2023; Accepted 12 August 2023

Theriogenology Wild 3 (2023) 100050
2
tool to detect pregnancy in multiple species of baleen whales [1,2,
33–37] and to estimate pregnancy and reproductive rates [4,7,38].
Testosterone is a major androgen in mammals; in the testis, where it
is primarily secreted and stimulates spermatogenesis. This androgen
plays an important role in the onset of sexual maturity for the devel-
opment of both primary and secondary sexual characteristics, [32] as
well as in the development of primary and secondary sexual character-
istics and behaviors [32,39–43]. Cetaceans are mostly seasonal breeders
with testosterone concentrations following a cyclic trend, peaking in
blubber before mating, then declining after breeding has occurred [44].
Seasonal trends in blubber testosterone have been reported in males
from the Pacic populations of blue (Balaenoptera musculus) and gray
(Eschrichtius robustus) whales [1,2] and in humpback whales (Megaptera
novaeangliae) [6,45]. In female whales, the role of testosterone and other
androgens have been investigated as potentially becoming primary
steroids during specic times in the reproductive cycle, such as prior to
parturition [46].
The present study evaluated progesterone and testosterone concen-
trations to conduct in-depth analyses on humpback whales found in the
waters near Juneau, Alaska. This subpopulation that uses the waters in
Southeast Alaska as their feeding grounds is part of the larger North
Pacic humpback whale population, with most whales (98 %) thought
to breed in the waters adjacent to the Hawaiian Islands, with a small
percentage (2 %) thought to breed in Mexican waters [47,48]. Hump-
back whales can be individually identied using photographs of
distinctive markings on their ventral ukes and dorsal ns; conse-
quently, photo-identication (i.e., photo-ID) of free-ranging whales is a
valuable research tool for tracking individuals through space and time
[49]. There are over 200 known individuals cataloged in the Juneau
waters (www.JuneauFlukes.org), many of which have high site delity
to the Juneau area and long-term sighting histories. Having access to
sighting histories for these whales provides a valuable context for
interpreting their reproductive physiology as well as for ongoing
monitoring and management. Juneau, Alaska, is a particularly impor-
tant area for research and monitoring given the thriving whale watching
industry in this community that relies on humpback whale presence.
With over 68 commercial whale watching boats running tours in a
relatively small tour area, within 25 km of Auke Bay from April into
October throughout the summer, Juneau’s whale watching industry is
estimated to generate over $60 million in direct economic revenue to the
local economy, while offering 367,000 cruise travelers a conduit to
experience humpback whales in their natural environment [50].
Research and monitoring are essential to ensure sustainability of the
industry and understand any potential impacts from tourism [51].
The North Pacic humpback whale population has been touted as a
recovery success as the Hawai’i distinct population segment increased
by more than 10-fold prior to the 2014–2016 North Pacic marine
heatwave and has experienced an estimated annual increase in the last
two decades of 6 – 7 % [52–54]. Such long-term increases in humpback
whale abundance generally result from both elevated reproductive rates
and increased survival [55–57]. In order to better understand repro-
ductive rates, several reproductive parameters are considered as
accepted norms for the North Pacic humpback whale population: the
breeding season is considered the winter months of November through
March, average annual reproductive rates are 1 calf every 2–3 years,
although annual calving is reported; 8–12 years is the typical age at rst
calving; and calf mortality rates are reported at 18–20 % [7,56,58–64].
In addition, male humpback whales are considered sexually mature at a
minimum age of 5 years [65,66] or when they reached the body lengths
of 14–15 m (46–49 ft) [67]. While numerous methods are available to
assess reproductive physiology, the use of endocrine methods has
blossomed with humpback whales [6,7,37,38,45,46,68]. For instance,
in the North Pacic population, blubber progesterone concentrations
were used to determine the number of presumed pregnant females [7,
68], and in turn applied to calculate pregnancy rates [7]. In other
populations of humpback whales, blubber progesterone concentrations
were also used in models to calculate pregnancy rates [37,38]. Other
studies have investigated testosterone and androstenedione as a marker
for the last phase of pregnancy and approaching parturition [46].
Additionally, studies on testosterone in males showed hormone con-
centrations to be lowest in the summer, followed by an increase in the
fall and a peak in the winter months [6,45].
The overall goal of the present study was to describe seasonal and
annual progesterone and testosterone proles in humpback whales
sampled in two consecutive years near Juneau, Alaska. This study was
unique in that repeated samples from both within the same year and
from consecutive years were purposefully targeted and collected from
individual whales with well-known life histories, whereas previous
studies that collected repeat samples were mainly accomplished with
opportunistic sampling. This difference provides more thorough
coverage of the whales, allows for the reproductive physiology to be
detailed at the level of individual whales, and therefore provides tighter
conclusions about the population and species. The specic objectives
were as follows:
1) To assess the mean and ranges of progesterone and testosterone
concentrations in the blubber of adult male and female humpback
whales in different reproductive states. This objective allows us to set
a specic threshold to determine pregnancy, to better understand
lactational anestrous, whales who are pregnant and lactating at the
same time, and the role of testosterone in females and juvenile males
relative to the timing of puberty.
2) To assess summer season pregnancy and calving rates for sexually
mature females, using annual sighting histories to conrm preg-
nancies with the presence of a calf the following year. This objective
enables the interpretation of pregnancy and calving rates, and the
difference between the two, based on resighting whales that were
predicted to be pregnant the previous year from elevated proges-
terone biomarkers. These data were also used to estimate an annual
population growth rate for this subpopulation.
3) To evaluate differences in seasonal residency rates according to sex
and reproductive state. With a measure of seasonal residency of
whales that spend the majority of the feeding season near Juneau,
Alaska, the data from the present study can be used to determine if
residency information is related to reproductive state.
2. Methods
2.1. Study site and samples collection
Humpback whales were studied in the waters near Juneau, Alaska
(Fig. 1). This area generally overlaps with the area used by the Juneau
whale watching industry during years where tourism was greatly
reduced due to the COVID-19 pandemic. The study area is known for an
abundance of Pacic herring (Clupea pallasii), the preferred prey of local
humpback whales [69–73]. Surveys for photo-id and biopsy sampling
were conducted over 31 days from 17 May to 10 October 2020 and over
24 days from 17 May to 2 November 2021. Biopsy samples were divided
into two arbitrary season bins: “early” summer, including all samples
collected May through July, and “late” summer, including samples
collected August through October. The early sampling bin reects a
post-migration period where the humpback whales have recently
returned from their breeding grounds, which coincidentally relates to a
mid-gestational phase. The late sampling bin reects the end of the
feeding season, which is also a pre-migration period before returning to
the breeding grounds. Biopsy samples were always carefully linked to
the photo-ID for each whale sampled. A total of 33 individual whales (N)
were biopsied, with 71 biopsy samples (n) collected over two years of
sampling. In 2020, 24 individuals were sampled and of these, 10 were
sampled more than once (N =24 whales, n =34 samples), while in
2021, biopsy samples were collected from 25 unique individuals, with
12 of them being sampled more than once (N =25 whales, n =37
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
3
samples). A total of 16 individuals (9 females and 7 males) were sampled
in both years. Behavioral reactions to biopsy sampling were scored after
Weinrich et al. [74] as none (n =16, 23 %), low (n =40, 56 %),
moderate (n =13, 18 %), strong (n =1, 1 %), or unknown (n =1, 1 %).
The most commonly observed reaction was a tail ick (n =30, 42 %; low
reaction), followed by no reaction (n =16, 23 %), a hard tail ick (n =
13, 18 %; moderate reaction), sink (n =6, 8 %; low reaction), accelerate
(n =2, 3 %; low reaction), inch (n =1, 1 %; low reaction), rise (n =1, 1
%; low reaction), and unknown (n =1, 1 %). The single reaction scored
as strong included a tail ick followed by two breaches.
Blubber samples for hormone analysis were collected from the ank
of each whale using a modied.22 caliber rie or a 150 lb. crossbow,
both of which launched an untethered projectile equipped with a sterile
stainless-steel biopsy dart tip and otation for retrieval [7]. The iden-
tication of each whale was determined through photo-ID prior to bi-
opsy. Samples were stored on ice or in the vessel’s freezer after
collection until they could be transported to the lab for processing.
While sex for some whales was known from long-term sighting re-
cords, sex for all individuals sampled was genetically determined from
excess biopsy tissue using previously established methods in the Oregon
State University’s Cetacean Conservation and Genomics Laboratory
[75]. A total of 38 samples (2020: n =19, 2021: n =19) were identied
as females and 33 as males (2020: n =15, n =18). Historic sighting
histories for biopsied whales came from unpublished Teerlink, Pearson,
and Moran databases and the Juneau Flukes Catalog (www. Juneau-
Flukes.org). Adult age class was determined using any combination of: i)
historic sighting records that could conrm a sighting history of ≥7
years (i.e., the average age of sexual maturation for North Pacic
humpback whales); ii) for females, the presence of a dependent calf; and
iii) eld evaluation based on size. Specically, length of sighting history
was used to determine age-class of all but seven whales (two females and
ve males); these individuals were identied as adult based on their
relative size compared to other whales and differentiated by experienced
eld practitioners.
2.2. Hormone extraction and analysis
For hormone analysis, the blubber portion of individual biopsies was
separated from the skin. Sample weights from the individual biopsies
ranged between 0.07 and 0.23 g (mean ±standard deviation =0.16
±0.03 g). Hormone extraction was performed following the step-wise
process described in Atkinson et al. [[4,7] adapted from Mansour
et al. [33] and Kellar et al. [76]. Briey, samples were rst macerated
manually using a Teon tip homogenizer in 1 ml of ethanol, then
extracted using a series of solvent-supernatant transfers, with multiple
organic solvents (ethanol, acetone, ether, acetonitrile and hexane).
Fig. 1. Map of the sampling area in Southeast Alaska and all biopsies collected in 2020 (red) and 2021 (blue). The square shapes represent adult male humpback
whales and the crosses represent adult female humpback whales.
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
4
Extracts were diluted in 1 ml of methanol before being aliquoted for
each assay, dried under forced air, and rehydrated with 250
μ
l of assay
buffer. Hormone concentrations were measured using enzyme immu-
noassays (EIA) for progesterone and testosterone from Enzo Life Science
(cat # ADI-900–011 and ADI-900–065), previously validated for this
species and tissue [7]. A standard curve and a zero binding (Assay
buffer) prepared according to the manufacturer protocol were run for
each plate. Specically, for the progesterone assay the standard con-
centrations ranged from 15.62 to 500 pg/ml with six points tting a
four-parameter logistic curve (4PLC) and for the testosterone assay the
standard curve was made of ve standards tted to a 4PLC ranging from
7.81 to 2000 pg/ml. Standards and samples were assayed in duplicate
and optical density was read at 405 nm using a TECAN M600
plate-reader. Samples were run at dilutions that allowed the percent
binding to fall on the linear section of the standard curve (30–70 %).
When sample percent binding fell outside this range, sample extracts
were concentrated or diluted appropriately and re-assayed. Addition-
ally, when the intra-assay coefcient of variation (CV) was above 15 %,
samples were also re-assayed. Inter-assay variation was assessed
through the calculations of three internal controls (ED 20, ED 50 and ED
80). The inter-assay CV for the three internal controls were 3.9 %, 9.5 %
and 7.5 % for the progesterone assay (n =4) and 3.7 %, 7.9 % and 6.8 %
for the testosterone assay (n =3).
2.3. Data Analysis
All biopsy samples used for analyses were from adult whales.
Reproductive state was assigned to each sample based on sighting his-
tory data and progesterone concentrations. Reproductive status was
dened using a standardized set of criteria (Table 1). Pregnancy was
conrmed if a presumed pregnant whale was sighted the following year
with a calf. For the present study, the lowest progesterone concentration
(i.e., 15.63 ng/g) from a conrmed pregnant whale (Animal ID 1583)
was chosen as the threshold for pregnancy. This arbitrary threshold falls
well within the uniform distribution identied by the model in Atkinson
et al. [7] using the same EIA system (Enzo LifeScience). All females with
progesterone concentrations equal or higher than 15.63 ng/g were
considered pregnant, while all females with progesterone concentra-
tions lower than 15.63 ng/g were considered resting (Table 1).
All statistical analyses for objectives 1 and 2 were performed in R
[77]. All graphs were made using the R packages ggplot2 [78] and
ggbreak [79]. For analysis, the values from the rst collected samples for
each individual from each year were compiled as a subset and analyzed
to conrm that progesterone concentrations were signicantly different
across female reproductive states and to evaluate difference in testos-
terone between adult males and females, and among females of different
reproductive states. Samples from the same individuals collected in
different years were considered independent. Because hormone con-
centrations tend to have a skewed distribution, data were
log-transformed. After graphically assessing normality distribution of
residuals using the function qqnorm and homogeneity of variances using
the Bartlett’s test, an ANOVA test followed by a Tukey post-hoc test was
performed on log-transformed progesterone and testosterone concen-
trations from females of different reproductive states. Log-transformed
testosterone concentrations were tested for differences between males
and females using a Student’s T-test.
Subsequently, concentrations were binned by “early” summer, for
samples collected between May and July (2020: n =10, 2021: n =19)
or “late” summer, for samples collected between August and October
(2020: n =24, 2021: n =18). The early sampling bin reects a post-
migration period where the humpback whales have recently returned
from their breeding grounds, which coincidentally relates to a mid-
gestational phase. The late sampling bin reects the end of the feeding
season, which is also a pre-migration period before returning to the
breeding grounds. Six individuals had samples in each bin for each year
(i.e., all four bins), seven individuals had samples in three of four bins,
ve individuals had samples in three of four bins, and the remaining 15
individuals had only one sample-bin combination. For three individuals
sampled in 2020 (Animal IDs 1703, 1783, 2070), their repeated samples
both fell into the late bin and thus only the rst value was included to
avoid pseudoreplication. To calculate a population growth rate from
these data, we used a Lotka model [80], which assumed calf survival of
0.8, calving rate from the present data, adult survival 0.97, recruitment
rate of 0.51. This model is used to provide an estimate of the annual rate
of population increase, which we applied to the whales in this study.
SPSS v. 28.0 was used for statistical analysis of objective 3. Alaska
residency rate was calculated to determine if residency information may
be related to reproductive state. The residency rate was calculated in
days as the timespan between the rst and last sighting of an individual
whale during a given eld season. Due to non-normal distributions,
differences in residency rate according to reproductive state were
analyzed with Kruskal-Wallis tests. The alpha level was set to 0.05.
3. Results
3.1. Objective 1: variation in reproductive hormones by sex and
reproductive state
Progesterone was detected in all 71 samples and testosterone was
detected in 70 of 71 samples, from 33 unique individuals. In females,
progesterone ranged from 0.03 to 44.61 ng/g and testosterone from
0.04 to 0.31. In adult males, progesterone concentrations ranged from
0.04 to 0.98 ng/g and testosterone from 0.05 to 2.0.
To conrm progesterone as a biomarker of pregnancy and to eval-
uate difference in testosterone between adult males and females, and
among females of different reproductive states, a subset of data were
made from the rst sample collected for each whale in each year. Pro-
gesterone concentrations were signicantly different among female
reproductive states (ANOVA test assuming unequal variances F=73.6,
df=3, p <0.0001), with pregnant and pregnant/lactating females hav-
ing signicantly higher progesterone concentrations than lactating and
resting females. For testosterone, adult males had signicantly higher
concentrations (Student’s T test: t = − 2.5, df=41.1, p =0.02). No sig-
nicant differences were found in testosterone concentrations among
females of different reproductive states (ANOVA: F=1.4, df=3,
p=0.29).
Mean and ranges of concentrations for each sex and reproductive
state for both progesterone and testosterone were calculated for samples
collected in early (May - July) and late (August - October) summer
(Tables 2 and 3). Progesterone concentrations clearly fell into two
groups (Fig. 2a): baseline (<1.0 ng/g progesterone) and elevated
(>15.6 ng/g). This separation did not occur for testosterone (Fig. 2b).
3.2. Objective 2: summer season pregnancy and calving rates
All females that were conrmed to be pregnant by the sighting of a
calf the following year had elevated progesterone. This was the case for
Table 1
Description of reproductive states for adult female humpback whales.
Reproductive
state
Description
Lactating Adult females that were sighted accompanied by a calf in the
year of sampling.
Pregnant Adult females that had a progesterone concentration >15.6 ng/
g. Most of these pregnancies were also conrmed by
documenting the mother with a calf in the following year.
Resting Adult females with known reproductive history and with a
progesterone concentration <lower than 3.1 ng/g (Atkinson
et al. 2023).
Pregnant/
lactating
Adult females that had a progesterone concentration >15.6 ng/
g and were sighted accompanied by a calf the year of sampling.
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
5
four of the ve pregnant females sampled in 2020 and for two of the four
pregnant females sampled in 2021. One sample in 2020 (SEAK 2006
prog =29.48 ng/g (early) and 23.17 ng/g (late) and two samples in
2021 (SEAK 1703 prog =20.49 (early) and 0.53 ng/g (late); SEAK 2147
prog=22.46 ng/g) had elevated progesterone but lacked sightings to
conrm pregnancy; however, they were presumed to be pregnant based
on progesterone concentrations that were above our 15.63 ng/g
threshold for pregnancy.
The pregnancy rate for sexually mature females determined during
the summer season from elevated blubber progesterone concentrations
was 0.45 (5 of 11 sexually mature whales), and 0.44 (4 of 9 mature
whales) for 2020 and 2021, respectively. The conrmed calving rate for
sexually mature females whose pregnancies were detected by proges-
terone concentrations in blubber biopsies and were subsequently
resighted with a calf the following year was 0.36 and 0.22 for 2020 and
2021, respectively; however, these numbers should be considered min-
imums as they did not include three whales whose pregnancies could not
be conrmed the following summer. Using the mean of these calving
rates (0.29), we calculated the annual rate of increase as 2.6 % for the
subpopulation of humpback whales in our study site.
Nine female whales had two samples collected within the same year,
both in 2020 and 2021. Progesterone and testosterone concentrations
were plotted for each individual, with the months of collection high-
lighted to provide a graphical representation of hormone concentrations
over a short (i.e., <6 months) temporal scale (Fig. 3). For all but one
female that were categorized as pregnant, progesterone remained
elevated. The exception was sexually mature female #1703, who was
sighted with a calf in 2020 and had a progesterone concentration of
20.5 ng/g for the rst sample collected in 2021, which was well above
the lowest concentrations of conrmed pregnant females. However, the
second sample collected from #1703 in 2021 showed a drastic drop in
this hormone concentration (0.5 ng/g). This individual was sighted on
eight different days in the waters surrounding Juneau during 2022 and
was never sighted accompanied by a calf. Another noteworthy adult
female whale is #1538, who was sighted accompanied by a calf in 2019,
2020, 2021 and 2022. Correspondingly, elevated progesterone concen-
trations were measured in samples from this animal collected in 2020
and 2021, and along with her resighting history conrms that she was
pregnant and returned to Alaska with a calf for four consecutive years.
3.3. Objective 3: differences in seasonal residency rates according to sex
and reproductive state
Mean residency rates were 72.9 d ±55.72 SD (range: 1–145, n=24)
in 2020 and 73.3 d ±60.15 SD (range: 1–170, n=25) in 2021 across all
sampled individuals (Fig. 4). Residency rate did not vary by reproduc-
tive state for 2020 (p =0.090) or 2021 (p =0.334), indicating that
residency is either not related to pregnancy or our sample size was too
small.
4. Discussion
Steroid hormones have been measured for decades as physiological
biomarkers in wildlife species, including large mysticete whales. Bio-
logical validation proles have been presented in multiple humpback
studies, conrming blubber progesterone as a good biomarker for
Table 2
Sample size of biopsies, mean and range of progesterone concentrations expressed in ng/g for males and females in different reproductive states for the two seasonal
bins (Early and Late summer).
2020
Early summer (May to July) Late summer (August to October)
Reproductive state Sample size Mean progesterone Range Sample size Mean progesterone Range
Adult males n=4 0.61 (0.26–0.97) n =11 0.43 (0.21–0.88)
Resting females n=1 0.38 n =1 0.5
Lactating females n=2 0.49 (0.29–0.68) n =4 0.36 (0.12–0.83)
Pregnant/Lactating females n=1 17.43 n =1 28.3
Pregnant females n=2 29.35 (29.29–29.48) n =4 33.16 (23.17–41.66)
2021
Early summer (May to July) Late summer (August to October)
Reproductive state Sample size Mean progesterone Range Sample size Mean progesterone Range
Adult males n=11 0.17 (0.04–0.52) n =7 0.23 (0.05–0.57)
Resting females n=3 0.06 (0.05–0.07) n =5 0.18 (0.03–0.53)
Lactating females n=2 0.25 (0.07–0.44) n =3 0.14 (0.13–0.15)
Pregnant/Lactating females n=1 24.92 n =1 15.63
Pregnant females n=3 21.78 (20.49–22.47) n =1 44.61
Table 3
Sample size of biopsies, mean and range of testosterone concentrations expressed in ng/g for males and females in different reproductive states for the two seasonal
bins (Early and Late summer).
2020
Early (May to July) Late (August to October)
Reproductive state Sample size Mean testosterone Range Sample size Mean testosterone Range
Adult males n=4 0.2 (0.05–0.44) n =11 0.25 (0.08–1.14)
Resting females n=1 0.13 n =1 0.07
Lactating females n=2 0.07 (0.06–0.07) n =4 0.06 (0.05–0.07)
Pregnant/Lactating females n=1 0.08 n =1 0.07
Pregnant females n=2 0.11 (0.09–0.14) n =4 0.11 (0.06–0.13)
2021
Early (May to July) Late (August to October)
Reproductive state Sample size Mean testosterone Range Sample size Mean testosterone Range
Adult males n=11 0.16 (0.07–0.42) n =7 0.67 (0.07–2.0)
Resting females n=3 0.09 (0.08–0.11) n =5 0.13 (0.06–0.31)
Lactating females n=2 0.12 (0.10–0.15) n =3 0.1 (0.06–0.12)
Pregnant/Lactating females n=1 0.17 n =1 0.09
Pregnant females n=3 0.13 (0.095–0.16) n =1 *
*
Not enough extract volume.
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
6
pregnancy [7,37,68]. Likewise, the measurement of testosterone in
blubber from humpback whales has also undergone validation [6,45].
However, few, if any, studies have the benet of working with a rela-
tively small subpopulation of whales with well-known individual
long-term sighting histories. The present study has conrmed the use of
progesterone to detect pregnancy and further established at the indi-
vidual level that annual breeders can be monitored and that an obligate
lactational anestrous does not exist for humpback whales. The study also
calculated pregnancy and calving rates using detailed resighting re-
cords, and was able to incorporate an annual population growth rate and
Fig. 2. A) Progesterone, and B) Testosterone concentrations in blubber samples from individual adult females collected in 2020 (red) and 2021 (blue). The x-axis
represents the whale ID. For progesterone concentrations, the dashed line represents the progesterone threshold for pregnancy (15.63 ng/g). All individuals were
resighted the following year either with (circles) or without (triangles) a calf.
Fig. 3. A) Progesterone, and B) Testosterone concentrations in adult females sampled up to 4 times through the 2020 (red) and 2021 (blue) sampling periods. Graphs
were created using the r package ggbreak (Chen et al., 2021).
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
7
the residency period of individual whales to better dene the conditions
of their period in the summer feeding grounds around Juneau, Alaska.
Satisfying these objectives has allowed an in-depth analysis of the
reproductive physiology at the level of individual whales, and therefore
provides tighter conclusions about the population and species.
The subpopulation of humpback whales in the present study pro-
vided a unique group of whales in that 1) over 200 individual humpback
whales have been cataloged in the Juneau area since 2006, including
some animals with high site delity to Juneau and some whales with
only a single sighting that transited the area; and 2) some individuals
have more than a decade of whale monitoring effort. Along with the
ability to collect purposefully repeated samples within a season, and
conduct a detailed study for over two years, these whales were an ideal
group of animals to further the study of reproductive physiology and
provide details at the individual level. The present study provided the
opportunity to observe short-temporal trends in progesterone concen-
trations in blubber tissues, as some individual females were sampled
more than once each year and/or in both 2020 and 2021. While the
sample size was limited, blubber progesterone concentrations did not
appear to increase over time in pregnant females, supporting previous
studies indicating a lack of seasonal trend in this hormone concentration
in multiple whale species. For instance, Clark et al. [68] did not nd
changes in progesterone concentrations in pregnant female humpback
whales sampled between the months of May and December off the coast
of California. Similarly, although with a limited sample size, Melica et al.
[1] did not nd any difference in hormone concentration in pregnant
blue whales sampled in the winter and summer grounds. However,
repeated samples in the present study were collected 1–3 months apart,
possibly too short of a time span to reect different gestational stages.
Because of the ability to conrm the results of a detected pregnancy
through the follow up of a calf sighting the following season, the present
study conrmed that not only are blubber progesterone concentrations
good biomarkers for pregnancy, but testosterone concentrations in fe-
males do not reect the pregnancy status during the summer season.
These results are consistent with a longitudinal study of two pregnant
humpback whales that did not show that testosterone had a dened or
discernable pattern throughout pregnancy [81]. Our results appear to
contradict the ndings of another previous study on this species [46]
that indicated androgens - specically, testosterone and androstenedi-
one - were good biomarkers of pregnancy [46]. However, that study was
conducted in pre-parturient humpback whales who were transiting back
to the breeding grounds to calve and breed and successful pregnancies
were not corroborated through calf sighting [46]. The Juneau subpop-
ulation of humpback whales is well known and pregnancy status could
be conrmed in nearly all cases by the return of the pregnant female
with a calf during the following season. Although testosterone was not a
good biomarker for the detection of pregnancy during mid-gestation, it
was informative for adult male humpback whales. Testosterone was
characteristically highest in adult males during both early and late pe-
riods. As was previously reported by Cates et al. [6] testosterone in male
humpback whales follows a predictable pattern for capital breeders,
with increasing concentrations in the fall prior to the breeding season,
stimulating the onset of spermatogenesis. In the present study, we
qualitatively saw a similar pattern with a much smaller sample size than
the previous study used.
Using resighting data from a small and discrete subpopulation of
known whales to conrm pregnancy provides advancements in the eld
of reproductive physiology by supplying a tool to begin to study the
difference between pregnancy rates and calving rates. Pregnancy rates
in the Juneau humpback whales were approximately 25 % lower than
Atkinson et al. [7] reported for the greater North Pacic population. This
is likely inuenced by two things: 1) sample sizes of the Juneau
humpback whales were considerably smaller than for the previous
study, which was largely due to the present study only covering two
years, and 2) the pregnancy rates for the present study were based on
conrmation of sighting the presumed pregnant female in the following
season with a calf, which equals the calving rate. The calving rate
documented from calf sightings is inherently biased downward as it only
provides the number of calves that survived their rst migration, not
accounting for fetal or perinatal mortalities, or failure to thrive during
migration. The true calving and pregnancy rates are likely much higher,
and both should be interpreted as minimum rates. Regardless, the
calving rates calculated here (0.36 in 2020 and 0.22 in 2021) are
consistent with consistent with the calving rates (0.30–0.32) for
humpback whales with calves returning to Glacier Bay National Park
and Preserve, which is also in Southeast Alaska [59].
Knowing the reproductive rate is a critical life history parameter in
calculating trends in abundance or population growth from life history
data. Also, knowing the current population growth rate relative to the
maximum growth rate for the species is a good indicator of population
status relative to carrying capacity (also commonly known as K). Given
humpback whale populations can grow at 6 % per year or greater [52,
82], population growth rates less than half of that rate are consistent
with a healthy population above the maximum net productivity level
(MNPL), which is an important management parameter under the US
Marine Mammal Protection Act (MMPA). Specically, any marine
mammal populations above MNPL are considered healthy or at optimum
levels. Thus, it is reasonable to infer that a subpopulation with a growth
rate of 2.6 %, such as the Juneau humpback whale subpopulation used
in the present study, are likely to be considered healthy under the
MMPA.
It is plausible that progesterone in blubber may not change signi-
cantly during pregnancy, unless in response to a rapid increase or
decrease in circulating concentrations. For instance, we observed one
individual (#1703) in which progesterone concentrations showed a 20-
fold decrease over a 3-month interval. This individual was not sighted
with a calf in 2022, and this hormone decrease could be indicative of a
dramatic change in circulating progesterone concentration associated
with reproductive failure. Decreased progesterone over a short time
interval was previously reported in fecal samples from blue whales [25].
In the blue whale study, fecal samples from an adult female collected a
few days apart showed a drastic decrease in progesterone concentra-
tions. Interestingly, that individual was never observed with a calf
despite being sighted consistently in the Gulf of California since the early
Fig. 4. Mean residency (days) for adult male and female humpback whales in a) 2020 or b) 2021. The females are separated by reproductive state. There was no
difference in residency rate by reproductive state.
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
8
1990 s [1,25]. These results add to the existing evidence that monitoring
sex steroids can provide valid insights to understand reproductive losses
that may occur during the course of gestation. In another study, high
concentrations of fecal progesterone were used to identify likely preg-
nant females; however, two of these females were resighted without calf,
suggesting they might have experienced pregnancy failure or calf
loss/death [83]. These kinds of results highlight the need to better un-
derstand reproductive losses that may occur during gestation or in the
perinatal period and the conditions that precipitate those scenarios,
which may include changes in prey resources in a given area.
Despite the changes in metabolic activity in animals of different life
history stages, including pregnancy and lactation, the humpback whales
in the present study did not have different lengths of residency in their
summer season feeding grounds near Juneau based on their reproduc-
tive status. In addition, mean residency rates were similar between 2020
and 2021. These data are likely inuenced by prey resources in the
summer feeding grounds, and herring are known to be the preferred
prey in this region [69–73]. Although prey populations may uctuate,
during the course of the study, herring was abundant and not thought to
be limiting in any way (Moran, unpubl. data). The nding of a lack of
differences in residency likely supports the evidence for a stable prey
base, at least during the time periods that the present study covered.
Nonetheless, better understanding of the relationship between length of
residency, prey status, and pregnancy warrants further study.
As with any study involving large migratory animals and analytical
procedures, a few caveats and cautions should be noted. First, our
samples sizes are not high enough to facilitate multivariate analysis (e.
g., controlling for the effects of year and season on hormone concen-
trations). However, our sample sizes are consistent with or exceeded
those reported in other large whale hormone studies [68,81]. Second,
while blubber may not be an ideal medium for investigating short-term
(hours to days) physiological response, it provides valuable information
to understand the overall health of the individual and is relatively easy
to obtain through standard biopsy sampling. Furthermore, multiple
types of analyses and biomarkers can be investigated in skin and blubber
tissue (e.g., genetics, stable isotopes, fatty acids, contaminants), maxi-
mizing a tissue sample’s use. However, it should also be noted that the
present study used an absolute threshold for detecting pregnancy, and
that if a different assay or extraction system is used, that threshold may
shift. Finally, it is important to acknowledge that collection of biopsies is
minimally invasive to the animal, while also avoiding the possibility of
environmental contamination or that from other whales that can hinder
studies using respiratory blow or feces. The specic effort to purposely
re-sample the same individuals over time provides unique and valuable
data that allows for expanded analyses at the level of individual whale
health.
In summary, we quantied blubber sex steroid hormones through
biopsy sampling and residency and pregnancy conrmations through
resightings in a small and well-known subpopulation of humpback
whales, which allowed for the calculations of pregnancy, calving and
annual population growth rates. The targeted sampling of desired in-
dividuals was viable and repeated sampling was accomplished with
multiple whales, with documented and generally minimal behavioral
responses. Having both well-established baseline hormone proles as
well as differences between whales in known sex and reproductive
groups sets the stage to be able to conduct long-term monitoring of a
unique population. The conrmation of pregnancy rates and comparison
of calving rates can be built on in the future to better understand
reproductive losses that occur during placental gestation. As a start, we
suggest that losses during gestation likely are elevated during and post
migration, and pregnancy and calving rates should be interpreted in the
context of migratory status and as minimums until additional studies on
reproductive losses can occur. Even if the sample size was limited, these
whales provided an excellent population for detailed studies of indi-
vidual health assessments of known humpback whales, something that is
not as easily conducted in areas with less historical sighting data. Using
individual physiological data to reect on annual population growth
rates is something that denes a good sentinel of the health of the ma-
rine ecosystem.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
We thank Doug DeMaster for his help with the annual population
rate calculations, and the volunteers who assisted in collecting resight-
ing information and biopsy samples, including Monica Brandhuber,
Taylor White, Lara Dzinich, Trinity Johnson, Rayne Billings, Annie
Masterman, and Chris Pearson. This study was funded by NOAA Fish-
eries’ Alaska Region, Protected Resources Division, through a grant to
the Pacic State Marine Fisheries Commission (#20-169G and #23-
035G), the Biomedical Learning and Student Training (BLaST) Program
through the National Institute of General Medical Sciences of the Na-
tional Institutes of Health under three linked awards number
RL5GM118990, TL4GM118992 and 1UL1GM118991, and the Idea
Network for Biomedical Research and Education (INBRE) through an
Institutional Development Award (IDeA) from the National Institute of
General Medical Sciences of the National Institutes of Health under
grant number P20GM103395 at the University of Alaska Fairbanks
(UAF). Data were collected under NMFS permit #20648 and UAF IACUC
protocol #1604256. The content of this publication is solely the re-
sponsibility of the authors and does not necessarily reect the ofcial
views of the NIH, NOAA, or the U.S. Department of Commerce. The
authors have no conicts of interest to report.
References
[1] V. Melica, S. Atkinson, D. Gendron, J. Calambokidis, F. Mueter, Blubber endocrine
proles provide insights into reproductive biology in blue whales from the eastern
North Pacic Ocean, Gen. Comp. Endocrinol. 310 (2021), 113830, https://doi.org/
10.1016/j.ygcen.2021.113830.
[2] V. Melica, S. Atkinson, J. Calambokidis, A. Lang, J. Scordino, F. Mueter,
Application of endocrine biomarkers to update information on reproductive
physiology in gray whale (Eschrichtius robustus), PLoS One 16 (2021), e0255368,
https://doi.org/10.1371/journal.pone.0255368.
[3] V. Melica, S. Atkinson, J. Calambokidis, D. Gendron, A. Lang, J. Scordino,
Naturally stressed? Glucocorticoid proles in blubber of blue and gray whales in
response to life history parameters, Mar. Mamm. Sci. 38 (2022) 1524–1548,
https://doi.org/10.1111/mms.12954.
[4] S. Atkinson, D. Gendron, T.A. Branch, K.L. Mashburn, V. Melica, L.E. Enriquez-
Paredes, et al., Pregnancy rate and biomarker validations from the blubber of
eastern North Pacic blue whales, Mar. Mamm. Sci. 36 (2020) 6–28, https://doi.
org/10.1111/mms.12616.
[5] K.A. Cates, S. Atkinson, A.A. Pack, J.M. Straley, C.M. Gabriele, S. Yin,
Corticosterone in central North Pacic male humpback whales (Megaptera
novaeangliae): Pairing sighting histories with endocrine markers to assess stress,
Gen. Comp. Endocrinol. 296 (2020), 113540, https://doi.org/10.1016/j.
ygcen.2020.113540.
[6] K.A. Cates, S. Atkinson, C.M. Gabriele, A.A. Pack, J.M. Straley, S. Yin, Testosterone
trends within and across seasons in male humpback whales (Megaptera
novaeangliae) from Hawaii and Alaska, Gen. Comp. Endocrinol. 279 (2019)
164–173, https://doi.org/10.1016/j.ygcen.2019.03.013.
[7] S. Atkinson, T.A. Branch, A.A. Pack, J.M. Straley, J.R. Moran, C. Gabriele, et al.,
Pregnancy rate and reproductive hormones in humpback whale blubber: dominant
form of progesterone differs during pregnancy, Gen. Comp. Endocrinol. 330
(2023), 114151.
[8] K.E. Hunt, R.M. Rolland, S.D. Kraus, Detection of steroid and thyroid hormones via
immunoassay of North Atlantic right whale (Eubalaena glacialis) respiratory vapor,
Mar. Mamm. Sci. 30 (2014) 796–809, https://doi.org/10.1111/mms.12073.
[9] K.E. Hunt, N.S. Lysiak, M. Moore, R.M. Rolland, Multi-year longitudinal proles of
cortisol and corticosterone recovered from baleen of North Atlantic right whales
(Eubalaena glacialis), Gen. Comp. Endocrinol. 254 (2017) 50–59, https://doi.org/
10.1016/j.ygcen.2017.09.009.
[10] R.M. Rolland, K.E. Hunt, S.D. Kraus, S.K. Wasser, Assessing reproductive status of
right whales (Eubalaena glacialis) using fecal hormone metabolites, Gen. Comp.
Endocrinol. 142 (2005) 308–317, https://doi.org/10.1016/j.ygcen.2005.02.002.
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
9
[11] E.A. Burgess, K.E. Hunt, S.D. Kraus, R.M. Rolland, Adrenal responses of large
whales: integrating fecal aldosterone as a complementary biomarker to
glucocorticoids, Gen. Comp. Endocrinol. 252 (2017) 103–110, https://doi.org/
10.1016/j.ygcen.2017.07.026.
[12] K.E. Hunt, R.M. Rolland, S.D. Kraus, S.K. Wasser, Analysis of fecal glucocorticoids
in the North Atlantic right whale (Eubalaena glacialis), Gen. Comp. Endocrinol. 148
(2006) 260–272, https://doi.org/10.1016/j.ygcen.2006.03.012.
[13] R. Rolland, W. McLellan, M. Moore, C. Harms, E. Burgess, K. Hunt, Fecal
glucocorticoids and anthropogenic injury and mortality in North Atlantic right
whales Eubalaena glacialis, Endanger. Species Res. 34 (2017) 417–429, https://doi.
org/10.3354/esr00866.
[14] K.E. Hunt, N.S. Lysiak, M.J. Moore, R.M. Rolland, Longitudinal progesterone
proles in baleen from female North Atlantic right whales (Eubalaena glacialis)
match known calving history, Conserv. Physiol. 4 (2016), cow014, 10.1093/
conphys/cow014.
[15] K.E. Hunt, R. Stimmelmayr, C. George, C. Hanns, R. Suydam, H. Brower, et al.,
Baleen hormones: a novel tool for retrospective assessment of stress and
reproduction in bowhead whales (Balaena mysticetus), Conserv. Physiol. 2 (2014),
https://doi.org/10.1093/conphys/cou030 cou030–cou030.
[16] R.M. Rolland, P.K. Hamilton, S.D. Kraus, B. Davenport, R.M. Gillett, S.K. Wasser,
Faecal sampling using detection dogs to study reproduction and health in North
Atlantic right whales (Eubalaena glacialis), J. Cetacea Res. Manag. 8 (2007) 121.
[17] S. Atkinson, D. Crocker, D. Houser, K. Mashburn, Stress physiology in marine
mammals: how well do they t the terrestrial model? J. Comp. Physiol. B 185
(2015) 463–486, https://doi.org/10.1007/s00360-015-0901-0.
[18] S. Atkinson, C. Combelles, D. Vincent, P. Nachtigall, J. Pawloski, M. Breese,
Monitoring of progesterone in captive female false killer whales, Pseudorca
crassidens, Gen. Comp. Endocrinol. 115 (1999) 323–332, https://doi.org/10.1006/
gcen.1999.7319.
[19] C.D. Champagne, N.M. Kellar, D.E. Crocker, S.K. Wasser, R.K. Booth, M.L. Trego, et
al., Blubber cortisol qualitatively reects circulating cortisol concentrations in
bottlenose dolphins, Mar. Mamm. Sci. 33 (2017) 134–153, https://doi.org/
10.1111/mms.12352.
[20] M.J. Keogh, S. Atkinson, Endocrine and immunological responses to
adrenocorticotrophic hormone (ACTH) administration in juvenile harbor seals
(Phoca vitulina) during winter and summer, Comp. Biochem Physiol. A Mol. Integr.
Physiol. 188 (2015) 22–31, https://doi.org/10.1016/j.cbpa.2015.06.011.
[21] T.R. Robeck, K.J. Steinman, J.K. O’Brien, Characterization and longitudinal
monitoring of serum androgens and glucocorticoids during normal pregnancy in
the killer whale (Orcinus orca), Gen. Comp. Endocrinol. 247 (2017) 116–129,
https://doi.org/10.1016/j.ygcen.2017.01.023.
[22] R.M. Rolland, K.M. Graham, R. Stimmelmayr, R.S. Suydam, J.C. George, Chronic
stress from shing gear entanglement is recorded in baleen from a bowhead whale
(Balaena mysticetus), Mar. Mamm. Sci. 35 (2019) 1625–1642, https://doi.org/
10.1111/mms.12596.
[23] R.M. Rolland, S.E. Parks, K.E. Hunt, M. Castellote, P.J. Corkeron, D.P. Nowacek, et
al., Evidence that ship noise increases stress in right whales, Proc. R. Soc. B: Biol.
Sci. 279 (2012) 2363–2368, https://doi.org/10.1098/rspb.2011.2429.
[24] K.J. Steinman, T.R. Robeck, J.K. O’Brien, Characterization of estrogens,
testosterone, and cortisol in normal bottlenose dolphin (Tursiops truncatus)
pregnancy, Gen. Comp. Endocrinol. 226 (2016) 102–112, https://doi.org/
10.1016/j.ygcen.2015.12.019.
[25] M. Valenzuela-Molina, S. Atkinson, K. Mashburn, D. Gendron, R.L. Brownell, Fecal
steroid hormones reveal reproductive state in female blue whales sampled in the
Gulf of California, Mexico, Gen. Comp. Endocrinol. 261 (2018) 127–135, https://
doi.org/10.1016/j.ygcen.2018.02.015.
[26] C.D. Champagne, N.M. Kellar, M.L. Trego, B. Delehanty, R. Boonstra, S.K. Wasser,
et al., Comprehensive endocrine response to acute stress in the bottlenose dolphin
from serum, blubber, and feces, Gen. Comp. Endocrinol. 266 (2018) 178–193,
https://doi.org/10.1016/j.ygcen.2018.05.015.
[27] N.M. Kellar, K.N. Catelani, M.N. Robbins, M.L. Trego, C.D. Allen, K. Danil, et al.,
Blubber cortisol: a potential tool for assessing stress response in free-ranging
dolphins without effects due to sampling, PLoS One 10 (2015), e0115257, https://
doi.org/10.1371/journal.pone.0115257.
[28] F.M.J. Mingramm, T. Keeley, D.J. Whitworth, R.A. Dunlop, Blubber cortisol levels
in humpback whales (Megaptera novaeangliae): a measure of physiological stress
without effects from sampling, Gen. Comp. Endocrinol. 291 (2020), 113436,
https://doi.org/10.1016/j.ygcen.2020.113436.
[29] M.R. Trana, J.D. Roth, G.T. Tomy, W.G. Anderson, S.H. Ferguson, Inuence of
sample degradation and tissue depth on blubber cortisol in beluga whales, J. Exp.
Mar. Biol. Ecol. 462 (2015) 8–13, https://doi.org/10.1016/j.jembe.2014.10.010.
[30] Iverson S.J., Koopman H.N. Blubber. Third edit. In: Wursig B, Thewissen JGM,
Kovacs KM, editors. Encyclopedia of Marine Mammals (Third Edition). Third edit.
Academic Press; 2019. pp. 107–110.
[31] Robeck T.R., O’Brien J.K., Atkinson S. Reproduction. Third Edit. In: Gulland F.M.D.
, Dierauf L.A., editors. CRC Handbook of Marine Mammal Medicine, Third Edition.
Third Edit. Boca Raton, FL: CRC Press; 2018. pp. 169–207.
[32] Atkinson S, Yoshioka M. Endocrinology of Reproduction. CRC Press. In: Miller DL,
editor. Reproductive Biology and Phylogeny of Cetacea: Whales, porpoises and
dolphins. CRC Press. CRC; 2007. pp. 171–192. doi:doi:10.1201/b10257-3.
[33] A.A.H. Mansour, D.W. Mkay, J. Lien, J.C. Orr, J.H. Banoub, N. ØIen, et al.,
Determination of pregnancy status from blubber samples in minke whales
(Balaenoptera acutorostrata), Mar. Mamm. Sci. 18 (2002) 112–120, https://doi.org/
10.1111/j.1748-7692.2002.tb01022.x.
[34] N.M. Kellar, J. Keliher, M.L. Trego, K.N. Catelani, C. Hanns, J.C. George, et al.,
Variation of bowhead whale progesterone concentrations across demographic
groups and sample matrices, Endanger. Species Res. 22 (2013) 61–72, https://doi.
org/10.3354/esr00537.
[35] E. Carone, M.A. Pardo, S. Atkinson, K. Mashburn, H. P´
erez-Puig, L. Enríquez-
Paredes, et al., Sex steroid hormones and behavior reveal seasonal reproduction in
a resident n whale population, in: K. Hunt (Ed.), Conserv. Physiol., 7, 2019,
https://doi.org/10.1093/conphys/coz059.
[36] C.T. Clark, A.H. Fleming, J. Calambokidis, N.M. Kellar, C.D. Allen, K.N. Catelani, et
al., Heavy with child? Pregnancy status and stable isotope ratios as determined
from biopsies of humpback whales, Conserv. Physiol. 4 (2016) cow050, https://
doi.org/10.1093/conphys/cow050.
[37] L. Pallin, J. Robbins, N. Kellar, M. Berube, A. Friedlaender, Validation of a blubber-
based endocrine pregnancy test for humpback whales, Conserv. Physiol. 6 (2018)
1–11, https://doi.org/10.1093/conphys/coy031.
[38] L. Pallin, C.S. Baker, D. Steel, N.M. Kellar, J. Robbins, D.W. Johnston, et al., High
pregnancy rates in humpback whales (Megaptera novaeangliae) around the Western
Antarctic Peninsula, evidence of a rapidly growing population, R. Soc. Open Sci. 5
(2018) 1–15 (Available), 〈http://rsos.royalsocietypublishing.org/content/5/5/
180017.abstract〉.
[39] J.C. Beehner, T.J. Bergman, D.L. Cheney, R.M. Seyfarth, P.L. Whitten, Testosterone
predicts future dominance rank and mating activity among male chacma baboons,
Behav. Ecol. Sociobiol. 59 (2006) 469–479, https://doi.org/10.1007/s00265-005-
0071-2.
[40] M.F. Bouissou, Androgens, aggressive behaviour and social relationships in higher
mammals, Horm. Res. 18 (1983) 43–61, https://doi.org/10.1159/000179778.
[41] H.M. Bryan, J.E.G. Smits, L. Koren, P.C. Paquet, K.E. Wynne-Edwards, M. Musiani,
Heavily hunted wolves have higher stress and reproductive steroids than wolves
with lower hunting pressure, Funct. Ecol. 29 (2015) 347–356, https://doi.org/
10.1111/1365-2435.12354.
[42] H.M. Bryan, C.T. Darimont, P.C. Paquet, K.E. Wynne-Edwards, J.E.G. Smits, Stress
and reproductive hormones in grizzly bears reect nutritional benets and social
consequences of a salmon foraging niche, PLoS One 8 (2013), e80537, https://doi.
org/10.1371/journal.pone.0080537.
[43] S.S. Negro, A.K. Caudron, M. Dubois, P. Delahaut, N.J. Gemmell, Correlation
between male social status, testosterone levels, and parasitism in a dimorphic
polygynous mammal, PLoS One 5 (2010) 1–8, https://doi.org/10.1371/journal.
pone.0012507.
[44] J.P. Schroeder, K.V. Keller, Seasonality of serum testosterone levels and sperm
density in Tursiops truncatus. J. Exp. Zool. 249 (1989) 316–321, https://doi.org/
10.1002/jez.1402490310.
[45] E.T. Vu, C. Clark, K. Catelani, N.M. Kellar, J. Calambokidis, Seasonal blubber
testosterone concentrations of male humpback whales (Megaptera novaeangliae),
Mar. Mamm. Sci. 31 (2015) 1258–1264, https://doi.org/10.1111/mms.12191.
[46] G. Dalle Luche, A.S.P. Boggs, J.R. Kucklick, J. Groß, D.W. Hawker, S. Bengtson
Nash, Androstenedione and testosterone but not progesterone are potential
biomarkers of pregnancy in Humpback Whales (Megaptera novaeangliae)
approaching parturition, Sci. Rep. 10 (1) (2020) 12, https://doi.org/10.1038/
s41598-020-58933-4.
[47] Martien K.K., Taylor B.L., Archer F.I., Audley K., Calambokidis J., Cheeseman T.,
et al. Evaluation of Mexico Distinct Population Segment of Humpback Whales as
units under the Marine Mammal Protection Act. 2021. NOAA-TM-NMFS-SWFSC-
658, 20 pgs.
[48] Wade P.R., Oleson E.M., Young N.C. Evaluation of Hawai ‘i distinct population
segment of humpback whales as units under the Marine Mammal Protection Act.
2021. NOAA Technical Memorandum NMFS-AFSC-430, 41 pgs.
[49] S. Katona, B. Baxter, O. Brazier, S. Kraus, J. Perkins, H. Whitehead, Identication of
humpback whales by uke photographs, Behav. Mar. Anim.: Curr. Perspect. Res. 3
(1979) 33–44.
[50] McDowell Group. Economic Analysis of Whale Watching Tourism in Alaska. 2020.
Prepared for NOAA. Available at 〈https://www.sheries.noaa.gov/resource/do
cument/economic-analysis-whale-watching-tourism-alaska〉.
[51] A.R. Schuler, S. Piwetz, J. Di Clemente, D. Steckler, F. Mueter, H.C. Pearson,
Humpback Whale Movements and Behavior in Response to Whale-Watching
Vessels in Juneau, AK, Front. Mar. Sci. (2019), 710 (Available), 〈https://www.
frontiersin.org/article/10.3389/fmars.2019.00710〉.
[52] Calambokidis J., Falcone E.A., Quinn T.J., Burdin A.M., Clapham P.J., Ford J.K.B.,
et al. SPLASH: Structure of populations, levels of abundance and status of
humpback whales in the North Pacic. Final report for Contract AB133F-03-RP-
00078. 2008;57.
[53] J. Barlow, J. Calambokidis, E.A. Falcone, C.S. Baker, A.M. Burdin, P.J. Clapham, et
al., Humpback whale abundance in the North Pacic estimated by photographic
capture-recapture with bias correction from simulation studies, Mar. Mamm. Sci.
27 (2011) 793–818.
[54] J.M. Mobley, S. Spitz, R. Grotefendt, P. Forestell, A. Frankel, G. Bauer, Abundance
of humpback whales in Hawaiian waters: Results of 1993-2000 aerial surveys, Rep.
Hawaii. Isl. Humpback Whale Natl. Mar. Sanctuary (2001) 9.
[55] Muto M.M., Helker V.T., Delean B.J., Young N.C., Freed J.C., Angliss R.P., et al.
Alaska marine mammal stock assessments, 2020. 2021.
[56] C.S. Baker, A. Perry, L.M. Herman, Reproductive histories of female humpback
whales Megaptera novaeangliae in the North Pacic, Mar. Ecol. Prog. Ser. 41
(1987) 103–114.
[57] Carretta J.V., Oleson E.M., Forney K.A., Muto M.M., Weller D.W., Lang A.R., et al.
US Pacic marine mammal stock assessments: 2021. 2022;25: A-389.
[58] C.M. Gabriele, J.M. Straley, J.L. Neilson, Age at rst calving of female humpback
whales in southeastern Alaska, Mar. Mamm. Sci. 23 (2007) 226–239, https://doi.
org/10.1111/j.1748-7692.2006.00100.x.
S. Atkinson et al.

Theriogenology Wild 3 (2023) 100050
10
[59] C.M. Gabriele, J.L. Neilson, J.M. Straley, C.S. Baker, J.A. Cedarleaf, J.F. Saracco,
Natural history, population dynamics, and habitat use of humpback whales over 30
years on an Alaska feeding ground, Ecosphere 8 (2017), e01641.
[60] J.M. Straley, Fall and winter occurrence of humpback whales (Megaptera
novaeangliae) in southeastern Alaska, Rep. Int. Whal. Comm. Spec. 12 (1990)
319–323.
[61] R. Cartwright, A. Venema, V. Hernandez, C. Wyels, J. Cesere, D. Cesere,
Fluctuating reproductive rates in Hawaii’s humpback whales, Megaptera
novaeangliae, reect recent climate anomalies in the North Pacic, R. Soc. Open
Sci. 6 (2019), 181463.
[62] C.M. Gabriele, J.M. Straley, S.A. Mizroch, C.S. Baker, A.S. Craig, L.M. Herman, et
al., Estimating the mortality rate of humpback whale calves in the central North
Pacic Ocean, Can. J. Zool. 79 (2001) 589–600.
[63] A.S. Craig, L.M. Herman, Habitat preferences of female humpback whales
Megaptera novaeangliae in the Hawaiian Islands are associated with reproductive
status, Mar. Ecol. Prog. Ser. 193 (2000) 209–216.
[64] Straley J.M., Gabriele C.M., Baker C.S., Engstrom D. Seasonal characteristics of
humpback whales (Megaptera novaeangliae) in southeastern Alaska. Proceedings of
the third glacier bay science symposium Anchorage: National Park Service. 1995.
pp. 229–238.
[65] R.G. Chittleborough, Marked humpback whale of known age, Nature 187 (1960)
164.
[66] C.M. Gabriele, C. Lockyer, J.M. Straley, C.M. Jurasz, H. Kato, Sighting history of a
naturally marked humpback whale (Megaptera novaeangliae) suggests ear plug
growth layer groups are deposited annually, Mar. Mamm. Sci. 26 (2010) 443–450.
[67] T.A. Jefferson, M.A. Webber, R.L. Pitman, U. Gorter, Marine Mammals of the
World: A Comprehensive Guide to Their Identication, Academic Press, 2015.
[68] C.T. Clark, A.H. Fleming, J. Calambokidis, N.M. Kellar, C.D. Allen, K.N. Catelani, et
al., Heavy with child? Pregnancy status and stable isotope ratios as determined
from biopsies of humpback whales, Conserv. Physiol. 4 (2016) 1–13, https://doi.
org/10.1093/conphys/cow050.
[69] S.M. Gende, M.F. Sigler, Persistence of forage sh ‘hot spots’ and its association
with foraging Steller sea lions (Eumetopias jubatus) in southeast Alaska, Deep Sea
Res. Part II: Top. Stud. Oceanogr. 53 (2006) 432–441.
[70] M.F. Sigler, D.J. Csepp, Seasonal abundance of two important forage species in the
North Pacic Ocean, Pacic herring and walleye pollock, Fish. Res 83 (2007)
319–331.
[71] M.F. Sigler, S.M. Gende, D.J. Csepp, Association of foraging Steller sea lions with
persistent prey hot spots in southeast Alaska, Mar. Ecol. Prog. Ser. 571 (2017)
233–243.
[72] K.M. Boswell, G. Rieucau, J.J. Vollenweider, J.R. Moran, R.A. Heintz, J.
K. Blackburn, et al., Are spatial and temporal patterns in Lynn Canal overwintering
Pacic herring related to top predator activity? Can. J. Fish. Aquat. Sci. 73 (2016)
1307–1318.
[73] J.M. Straley, J.R. Moran, K.M. Boswell, J.J. Vollenweider, R.A. Heintz, T.J. Quinn
II, et al., Seasonal presence and potential inuence of humpback whales on
wintering Pacic herring populations in the Gulf of Alaska, Deep Sea Res. Part II:
Top. Stud. Oceanogr. 147 (2018) 173–186.
[74] M.T. Weinrich, R.H. Lambertson, C.R. Belt, M.R. Schilling, H.J. Iken, S.E. Syrjala,
Behavioral reactions of humpback whales Megaptera novaeangliae to biopsy
procedures, Fish. Bull. 90 (1992) 588–598.
[75] C.S. Baker, D. Steel, J. Calambokidis, E. Falcone, U. Gonz´
alez-Peral, J. Barlow, et
al., Strong maternal delity and natal philopatry shape genetic structure in North
Pacic humpback whales, Mar. Ecol. Prog. Ser. 494 (2013) 291–306.
[76] N.M. Kellar, M.L. Trego, C.I. Marks, A.E. Dizon, Determining pregnancy from
blubber in three species of delphinids, Mar. Mamm. Sci. 22 (2006) 1–16, https://
doi.org/10.1111/j.1748-7692.2006.00001.x.
[77] R Core Team. R: A Language and Environment for Statistical Computing. R
Foundation for Statistical Computing. Vienna, Austria, Austria: R Foundation for
Statistical Computing, 2021. doi:〈10.1007/978-3-540-74686-7〉.
[78] H. Wickham, W. Chang, M.H. Wickham, Package ‘ggplot2.’, Creat. elegant data
Vis. Using Gramm. Graph. Version 2 (2016) 1–189.
[79] S. Xu, M. Chen, T. Feng, L. Zhan, L. Zhou, G. Yu, Use ggbreak to effectively utilize
plotting space to deal with large datasets and outliers, Front. Genet. 12 (2021)
2122.
[80] DeMaster D.P. Considerations regarding an estimation of Rmax for Hector’s and
Maui dolphins. Report of. Report of the Workshop on Hector’s and Maui Dolphins
in New Zealand: Consideration of Spatial Risk Assessment of Threats. Report of.
2023. pp. 20–25. SC/69A/REP/04.
[81] C.L. Lowe, K.E. Hunt, J.L. Neilson, C.M. Gabriele, S.S. Teerlink, C.L. Buck,
Reproductive steroid hormone patterns in baleen of two pregnant humpback
whales (Megaptera novaeangliae), Integr. Comp. Biol. 62 (2022) 152–163.
[82] J. Barlow, P.J. Clapham, A new birth-interval approach to estimating demographic
parameters of humpback whales, Ecology 78 (1997) 535–546.
[83] K.E. Hunt, J. Robbins, C.L. Buck, M. B´
erub´
e, R.M. Rolland, Evaluation of fecal
hormones for noninvasive research on reproduction and stress in humpback whales
(Megaptera novaeangliae), Gen. Comp. Endocrinol. 280 (2019) 24–34, https://doi.
org/10.1016/j.ygcen.2019.04.004.
S. Atkinson et al.