Technical Reports of the Institute of Cetacean Research (2018)

pp. 56–63

Technical Report (not peer reviewed)

Results of feasibility studies on novel non-lethal techniques toaddress the main objectives of NEWREP-A
Mutsuo Goto* and Satoko Inoue
Institute of Cetacean Research, 4–5 Toyomi-cho, Chuo-ku, Tokyo 104–0055, Japan*Contact e-mail: goto@cetacean.jp

ABSTRACTThis paper presents the progress made in the research conducted to evaluate the feasibility of some novel
non-lethal techniques. The techniques are DNA methylation for age determination and progesterone analysis
for determining the reproductive status of whales. Age and reproductive status of whales are key information
for addressing one of the main objectives of the NEWREP-A. Potentially both techniques can be used on biopsy
samples collected from free ranging whales. Regarding the former technique, eﬀort was spent to investigate
diﬀerences in methylation rates between Antarctic minke and humpback whales. Regarding the latter technique, eﬀort was made to examine progesterone from blubber of female Antarctic minke whales for whichhigh qualitative reproductive data was already available. A ﬁnal evaluation of both techniques will be made in
the near future considering the protocol developed for evaluating novel non-lethal techniques (Mogoe, thisissue).

INTRODUCTION
The Institute of Cetacean Research (ICR) conducts whale
research using both lethal and non-lethal techniques. Lethal techniques are required for key research objectivesof NEWREP-A such as the estimation of biological parameters based on age and reproductive data, and feeding
ecology based on qualitative and quantitative analyses ofstomach contents.
Routine non-lethal techniques used by ICR include systematic sighting surveys for abundance estimates, oceanographic surveys for ecological studies and photo-id andbiopsy sampling for studies on movement, distribution
and stock structure in blue, humpback and right whales.
These routine non-lethal techniques have been used by
ICR for many years in its whale research programs in the
Antarctic and western North Paciﬁc.
More recently some novel non-lethal techniques havebeen proposed, which potentially could be used to address some key objectives of NEWREP-A. For example,
DNA methylation in skin to investigate the age of theanimals, analysis of progesterone level in blubber to investigate the reproductive status of female whales, andstable isotopes in skin/blubber to study the feeding ecology of whales. These techniques can potentially be usedwith skin/blubber samples obtained by biopsy sampling.
ICR started the evaluation of the feasibility of using the56

non-lethal techniques mentioned above. The evaluation
involves two aspects, the ﬁrst is whether biopsy sampling(source of the samples) is feasible in the target species of
the research programs. The other aspect is the analyticalone, which involves laboratory and statistical analyses(see Mogoe, this issue).
The degree of diﬃculty of biopsy sampling is diﬀerent
among whale species, being more diﬃcult for smallerand fast swimming species such as the minke whale. Biopsy sampling was considered feasible in the case of large
whales such as sei and Brydeʼs whales in the North Paciﬁc
however it was considered not feasible for the case of
North Paciﬁc common and Antarctic minke whales (Yasunaga et al., 2017; 2018).
This paper summarizes the laboratory and statisticalprocedures to investigate the feasibility of using nonlethal techniques to determine age and reproductive status in Antarctic minke whale, the target species for lethal
sampling under the NEWREP-A. Tissue samples from thesampled Antarctic minke whale individuals were used
as a proxy for biopsy sampling of skin and blubber. The
outputs of these studies will be used for a ﬁnal evaluationof non-lethal techniques based on some speciﬁed criteria(see Mogoe, this issue).

Results of feasibility studies on novel non-lethal techniques to address the main objectives of NEWREP-A

DNA METHYLATION FOR AGE DETERMINATION
IN WHALES
DNA methylation as a tool for age determination
Chronological age is an important factor in animal ecology
because many biological characteristics change with time
(Jarman et al., 2015). Biological ageing is a combinationof programmed processes (Berdasco and Esteller, 2012;
Horvath, 2013) and accumulated changes caused byunrepaired environmental damage (Kujoth et al., 2005).
Recent evidence suggests that epigenetic changes areboth directing the process of ageing and being caused byit (Maegawa et al., 2010; Koch et al., 2011; Winnefeld andLyko, 2012; Hannum et al., 2013; Horvath, 2013).
The best studied class of epigenetic change in vertebrates is the methyl group presence or absence at the C5
position of Cytosine residues that are adjacent to Guanidine residues (ʻCpG sitesʼ) (Figure 1). CpG methylationlevels play an important role in control of gene expression, where higher methylation levels (ʻhypermethylationʼ) generally reduce gene transcription rate.
Methylation changes at speciﬁc CpGs have been
linked to age in mice (Maegawa et al., 2010) and humans (Christensen et al., 2009; Grönniger et al., 2010;
Bocklandt et al., 2011; Koch and Wagner, 2011; Hannum
et al., 2013). For the cetacean species, the DNA methylation approach was recently developed and applied to
humpback whales with the aim of age determination inthis species (Polanowski et al., 2014).

ed DNA, which was used in the case of the humpback
whale (Polanowski et al., 2014) and in the present study
for Antarctic minke whales. This method implies the useof bisulﬁte treatment of DNA to determine its patternof methylation. In animals it predominantly involves the
addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicatedin repression of transcriptional activity. Treatment of DNA
with bisulﬁte converts cytosine residues to uracil, but
leaves 5-methylcytosine residues unaﬀected. Therefore,
DNA that has been treated with bisulﬁte retains only
methylated cytosines. The percentage of DNA mismatchis recorded, this gives the user a percentage methylation
per CpG site.
Research on DNA methylation at ICR
As a ﬁrst step to evaluate the DNA methylation for age
determination purpose, the same method used for
humpback whale was applied to Antarctic minke whale.
The purpose in this ﬁrst step was to compare DNA methylation rates between two baleen whale species. The
work conducted on Antarctic minke whale is explained in
the sections below.
Samples
A total of 100 Antarctic minke whale samples taken during the JARPAII were used for the study. Each sample
had good age information obtained by earplug reading.
Details of the samples are shown in Table 1. DNA was extracted from each sample using standard protocols.

Detection methods
DNA methylation can be detected by several methods,
one of them being the pyrosequencing of bisulﬁte treat-

Figure 1.

Methylation of cytosine to form 5-methylcytosine occurs at the same C5 position on the pyrimidine ring
(upper right), the same position distinguishes thymine from the analogous RNA base uracil after bisulﬁteconversion (lower right), and then methylation rate is estimated by the ratio of thymine and cytosine.
57

Technical Reports of the Institute of Cetacean Research (2018)
Table 1The sample sizes of Antarctic minke whales used for DNA methylation analysis by pyrosequencing method by sampling season,sex and age classes.
SeasonSex

2010/2011

F
M
F
M

2011/2012
Total

Figure 2.

–10 11–20 21–30 31–40 41–

Total

10
7
1
1

16
8
8
3

9
8
11
1

3
2
6
1

3
1
1

41
25
27
7

19

35

29

12

5

100

PYROMARK 24 Pyrosequencing System used in thisstudy.

Figure 3.

58

Age

Identiﬁcation of age-related epigenetic markers in
Antarctic minke whale
The procedure for identiﬁcation of age-related DNA
methylation sites and measurement of methylation levels
followed Polanowski et al. (2014). The following sevenCpG sites in three genes were selected for this study because they showed signiﬁcant correspondence between
CpG methylation levels and age in humpback whales.
Gene TET2 (three sites: CpG+16; CpG+21 and CpG+31)
Gene CDKN2A (three sites: CpG+297; CpG+303 andCpG+309)
Gene GRIA2 (one site: CpG+202)
Measurement of cytosine methylation levels and correlation with age
Cytosine methylation levels were measured with Qiagen PyroMark assays. The pyrosequencing assays were
designed using PYROMARK Assay Design Software (Version 2.0.1, Qiagen). Pyrosequencing was performed on
a PYROMARK 24 Pyrosequencing System (Qiagen) (Figure 2). The PYROMARK Q24 software gave percentage
methylation values for each CpG site (Figure 3).
DNA methylation rates in the seven sites were scoredsuccessfully, and regressions of each CpG methylation
rate against age determined by earplug reading weremade (Figure 4).
Correlations of all seven CpG sites in three loci withage based on earplug reading were lower than in the caseof the humpback whale study (Table 2). The standard
deviation for Antarctic minke whale by the Leave One

An Example of output of CpG Pyrogram report by PYROMARK 24 Pyrosequencing System of three CpG
sites in TET2 gene. Methylation rate of each CpG site is estimated by the ratio of the integral of Thymine
to Cytosine in gray shadow.

Results of feasibility studies on novel non-lethal techniques to address the main objectives of NEWREP-A

Figure 4.

Regressions between CpG methylation and age determined by earplug at seven sites (n=100). CpG sites
are: (A) GRIA2_CpG+202, (B) TET2_CpG+16, (C) TET2_CpG+21, (D) TET2_CpG+31, (E) CDKN2A_CpG+297,(F) CDKN2A_CpG+303 and (G) CDKN2A_CpG+309.

Table 2Comparison of the coeﬃcient of determination (R2) between
methylation rates of seven CpG sites and age determined from
earplug reading in humpback (Polanowski et al., 2014) andAntarctic minke (this study) whales.
Gene and CpG position

Humpback whale

This study

GRIA2_CpG+202
TET2_CpG+16
TET2_CpG+21
TET2_CpG+31
CDKN2A_CpG+297
CDKN2A_CpG+303
CDKN2A_CpG+309

0.469
0.174
0.189
0.409
0.409
0.211
0.344

0.01610.06040.06830.1874>0.0001
0.01480.0784Out Cross Validation (LOOCV) method was estimated at
8.865 years (Goto et al., 2018), while that for the humpback whale was estimated at 2.991 years using the same
method (Polanowski et al., 2014). Therefore the main
conclusion of this study is that the methylation rates fora same gene and sites are diﬀerent between the two baleen whale species.
Future works
The ICR is planning investigation of additional informative methylation sites. Such investigation should include
analyses on how the methylation rates changes among
body tissues as well among body parts of the whale,
among other factors.
With all information at hand the feasibility of using the
DNA methylation technique for age determination in Ant59

Technical Reports of the Institute of Cetacean Research (2018)arctic minke whale can be further evaluated.

HORMONE ANALYSIS FOR DETERMINATION OF
REPRODUCTIVE STATUS IN WHALES
Progesterone analysis for determination of reproductive
status in female whales
In cetacean species female reproductive status has been
determined by direct observation of reproductive organs
such as ovaries and uteruses. Progesterone is one of thesex hormones produced from ovaries and placenta (whenan individual gets pregnant) and it has role in ovulationand maintaining pregnancy.
Previous studies suggested that an elevation in theconcentration of progesterone in serum can be indicativeof ovulation and pregnancy diagnosis in captive cetaceans (Sawyer-Steﬀan et al., 1983; Schroeder and Keller,
1990; Kirby, 1990). Recently, the relationship between
progesterone and reproductive status has been studied
based on samples which potentially can be obtainedby non-lethal approach from free ranging cetaceans(Table 3). Several of those studies reported that progesterone can be detected from the samples used, and thatits concentration was substantially higher in pregnant
than immature females.
Methods
There are two main groups of methods to measure progesterone level, one is liquid chromatography (e.g. HPLC
and LC-MS in Table 3) and the other is immunoassay. The
latter has been used in the studies conducted by the ICR.
Immunoassay
Immunoassay is a method that measures the presenceor concentration of target analytes (like hormones) in
solution using antigen-antibody reactions. The antigenantibody reaction is a speciﬁc binding between an anti-

gen and an antibody. The reaction can be used for thedetection of antigens or antibodies speciﬁc for analytes.
Immunoassay measures the formation of antigenantibody complexes by labeling or labeling-free format.
There are several types of immunoassays depending onlabeling material, for example, labeling by radioisotope
is called radioimmunoassay (RIA), labeling by enzyme iscalled enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). The immunoassay used at
the ICR is the latter one (ELISA) (Figure 5).
Research on progesterone at ICR
In the ICR, the relationship between concentration ofprogesterone in blubber and reproductive status in the
Antarctic minke whale was investigated by examining
female Antarctic minke whales. The main question waswhether or not progesterone concentration in blubber
can be used as an indicator of the reproductive status offemale whales.
Samples
Antarctic minke whales used in this study were collected
between December 2015 and February 2016 by NEWREPA in the Paciﬁc sector of the Antarctic Ocean. Whales
were sampled randomly from a predetermined zigzag
track line designed to cover the whole research area. A
total of 333 Antarctic minke whales (103 males and 230
females) were sampled during the survey. All sampled
females (230) were used in this study. The blubber tissues were obtained from the lateral side of each whale byresearchers on board the research base ship. The tissues
were stored at −20°C until analyses at the laboratory.
Determination of reproductive status
Reproductive status was determined by standard procedures (Lockyer 1984; 1987). The sexual maturity of

Table 3
Summary of the non-lethal studies to measure sex hormones in whales.
Species
MysticetiBlue whale Balaenoptera musculus
North Atlantic right whale Eubalaena glacialis
Humpback whale Megaptera novaeangliae

Odontoceti
Beluga Delphinapterus leucas
60

Samples

Methods

References

Feces
Feces
Blow
Blubber
Blubber
Blow

RIA, HPLC
RIA
LC-MS
EIA
EIA
LC-MS

Valenzuela-Molina et al. (2018)
Corkeron et al. (2017)
Hogg et al. (2009)
Clark et al. (2016)
Pallin et al. (2018)
Hogg et al. (2009)

Blow

ELISA

Richard et al. (2017)

Results of feasibility studies on novel non-lethal techniques to address the main objectives of NEWREP-A

Figure 5.

Schematic representation of ELISA. An antibody speciﬁc for a target protein is immobilized on the surfaceof microplate wells. Then it is incubated with samples containing the target protein and a known amount of
enzyme-labeled target protein. After the reaction, the substrate is added and the activity of the microplate
well-bound enzyme is measured. When the antigen level in the sample is high, the level of antibody-boundenzyme-labeled antigen is lower and the color is lighter. Conversely, when it is low, the level of antibodybound enzyme-labeled antigen is higher and the color is darker.

Figure 6.

Flow from collection of blubber sample to measurement of progesterone concentration.

females was determined by the presence of at least one
corpus luteum or corpus albicans in either ovary. In thecase where no corpus luteum and corpus albicans wasobserved, the female was categorized as immature. Reproductive status of mature female whales was classiﬁed
into three categories (resting, ovulating and pregnant),
based upon observation of the ovary and uterus.
Extraction and measurement of progesterone
The procedures for progesterone extraction and defatting treatment were performed according to Kellar et al.
(2013) and Nagata et al. (1996), respectively. Progesterone concentrations were determined by a commerciallyavailable ELISA kit, using the Crocodile Mini Workstation(Titertek Berthold, Germany). All samples were processed and quantiﬁed in triplicate. Figure 6 shows thesteps followed in the progesterone concentration study.
Results
Table 4 shows the results of the reproductive status determined by direct observation of reproductive organs,progesterone concentrations by reproductive status andother biological data for the total sample of 230 female
whales.
Seventy-six percent of the samples were mature females, and 90% of the mature females were pregnant.
About 73% of the immature whales were below the detection limit of the assay for progesterone concentration(0.2 ng/g). The pregnant females had the highest medianof 72 ng/g (range: 13–740 ng/g), with wider concentrations of progesterone. All pairwise comparisons apartfrom the ovulating-pregnant comparison, resulted in61Technical Reports of the Institute of Cetacean Research (2018)
Table 4
Biological data, determined reproductive status and progesterone concentration in the female Antarctic minke whales.
Immature

Resting

OvulatingPregnant

Body length (m)Median
7.37
8.81
9.01
8.78
Range
5.17–8.51 7.65–9.32 8.45–9.40 7.71–10.06
Ovary
total CL
Mean
0
0
1
1
Range
0
0
1–1
1–2
total CA
Mean
0
18
16
13
Range
0
1–44
0–45
0–40
Progesterone Concentration (ng/g)
Median
<0.2
15
26
72
Range
<0.2–2.6 <0.2–34
24–150
13–740
N
56
11
6
157

signiﬁcant statistical diﬀerences (Steel-Dwass post-hoc
test, p<0.05). However, overlap of progesterone concentrations was observed between each reproductive status
with the exception of the cases immature/ovulating andimmature/pregnant.
Summary
In this study, signiﬁcant diﬀerences were found in medianprogesterone concentration between all reproductive
categories except in the case between ovulating andpregnant females. However, the ranges of progesterone
concentration overlapped between each reproductive
category with the exception of the cases immature/ovulating and immature/pregnant. The results of this study
indicated that the progesterone concentration in blubber,which potentially can be obtained by biopsy sampling,cannot be used as an accurate diagnostic index to determine the reproductive status in the Antarctic minke
whale.

CONCLUDING REMARKS
Progress has been made in the work to evaluate novel
non-genetic techniques that potentially could be used toaddress some important objectives of NEWREP-A. The
degree of progress is diﬀerent among the speciﬁc technique examined and obviously further reﬁnement work
is required. Stable isotopes are also being examined toelucidate the feeding habits of some large baleen whale
species in the Antarctic. As stated earlier a ﬁnal evaluation of the feasibility of those techniques will be madein the near future following speciﬁc criteria (see Mogoe,
62

this issue).

ACKNOWLEDGEMENTS
We thank crew members and researchers that participated in the 2010/11 and 2011/12 JARPAII, 2015/16 and2016/17 NEWREP-A surveys in Antarctic for collecting
data and samples of the Antarctic minke whale used inthis study. We thank Natsumi Endo (Tokyo University ofAgriculture and Technology) for providing ELISA assay.

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