Online Early Version
A study on the improvement of age
estimation in common minke whales using
the method of gelatinized extraction of
earplug
Hikari Maeda1,*, Tadafumi Kawamoto2, and Hidehiro Kato1
1

Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 1088477, Japan
2
Radioisotope Research Institute, Tsurumi University School of Dental Medicine, 2-1-3
Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan*Corresponding Author: Hikari Maeda, E-mail: hikarim@affrc.go.jp; Current
address: National Research Institute of Far Seas Fisheries, 2-12-4 Fukuura, Kanazawa,
Yokohama 236-8648, Japan

ABSTRACT
We attempted to settle the potential problems of bias caused by too soft earplugs and poor
formation of the growth layers in age readings of common minke whales. Thus, we
examined the feasibility of a new technique of incorporating gelatin in order to collectearplugs for age assessment. Frozen sectioning and histology of the earplug core were also
used as methods to improve age estimation. Earplugs were collected by filling the space inthe external auditory meatus with gelatin, hardening the gelatin, earplug and its fragments,
by spraying with cooling gas, and removing the earplug embedded in gelatin. In 174 trials
with common minke whales in the Western North Pacific of coastal waters of Japan in
2007–2009, it was revealed that embedding earplugs with gelatin minimized breakage andprotected the neonatal line (NL). This method was particularly effective in younger animals.
As a result, the readability was improved. We also examined the histological sections,which were sliced using the Kawamoto specialized frozen sectioning technique, and stained
them separately with toluidine blue, haematoxylin and eosin, Sudan III, Sudan VII, andalizarin red S to display a clearer core surface image of the growth layers. The histologicalsections stained with alizarin red S provided the clearest images, in which we could easily
identify both dark and pale laminations. This suggested a close relationship with theseasonal changes in calcium intake from feeding. Earlier age estimation methods focusedon fat content in the growth layers; however, we found potential for an improvement in thereadability of unclear growth layers when focusing on calcium.

INTRODUCTION
Animal age is important basic information for population studies. Earplugs
were examined as an age character for the first time in baleen whales by
Purves (1955). Even today, earplugs are widely used as an age character(Gabriele et al. 2010, Lockyer 1984a, Nielsen et al. 2012). As shown in
Figure 1, the earplug accumulates in the external auditory meatus (Lillie
Maeda, H., Kawamoto, T., and Kato, H. (2013) A study on the improvement of age
estimation in common minke whales using the method of gelatinized extraction of
earplug. NAMMCO Scientific Publications. doi: http://dx.doi.org/10.7557/3.2609

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Maeda et al. (2013) Online Early Version

1910) and consists of a core and outer covering (Purves 1955, Ichihara
1959). The outer covering is secreted by epithelial cells in the external
auditory meatus, whereas, the core, comprising concentric light and darklaminae, is secreted by papillae on the surface of the glove finger (Purves
1955). Fat content tends to be lower in the dark layer and higher in the lightlayer from histological observations of fin whale (Balaenoptera physalus)
earplugs (Roe 1967). The light and dark layers are formed during thefeeding and breeding periods, respectively (Roe 1967). Since many baleen
whales migrate between a breeding place in low-latitude waters (winter) anda feeding place in high-latitude waters (summer), which is approximated byan annual cycle, such a living cycle is reflected as a growth layer; therefore,
one growth layer represents 1 year (Best 1982, Ohsumi 1964, Gabriele et al.
2010, Lockyer 1972; 1984b, Roe 1967). Besides, the neonatal line (NL)
which is created at birth (Ichihara 1964), is formed at the apical portion of
the core, and germinal layers which are newly created, are formed at thebasal portion (Fig. 2).

Fig. 1. Illustration of the ventral surface of the skull of a common
minke whale, and a magnified view (below) of an exposed external
auditory meatus. The ruler is graduated in millimeters.

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Age estimations from earplugs have been conducted in several species ofbaleen whales (Lockyer 1984a). However, earplugs of the common minke
whale (Balaenoptera acutorostrata) are generally believed to poorly reflect
the age because of their softness and poor formation of growth layers(Sergeant 1963, Christensen 1981, Christensen et al. 1990, Kato 1992,Auðunsson et al. 2013). Improving the age estimation rate and acquiring
more accurate age information are necessary for analyses, such as age
dynamic studies. Thus, it is necessary to examine the causes for the low age
estimation rate. There are two main factors that lower the age estimation
rate of common minke whales using earplugs. The first factor is breakage
during the collection stage. Most earplugs, particularly those of younger
animals, are easily damaged during the collection because of their softness
and small size. The second factor is the presence of earplugs with unclear
layering patterns in which it is difficult to identify the growth layers using
the existing age estimation method (Maeda and Kato 2012).
Traditionally, earplugs have been collected by exposing and cutting theexternal auditory meatus and then directly obtaining the internal earplug by
using tweezers. However, it is very difficult to collect earplugs in theirperfect condition by employing the existing method.

Fig. 2. Bisected surface of an earplug of a common minke whale. a:
Outer covering, b: total earplug length, c: core length. Scale bar: 5 mm

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Previously, Ichihara (1959) and Roe (1967) tested several staining solutions(Heidenhain’s iron haematoxylin, haematoxylin and eosin, Azan, Dopa
reagent, methyl green-pyronin (MGP), Schiff’s reagent, Sudan III and
Sudan Black) using frozen earplug sections. They found that Sudan III,
which detects fat cells, was the most useful staining solution for earplug
growth layers. Subsequently, there have been few earplug studies using
these histological methods.
In the present study, we describe a method to minimize the damage done to
earplugs at the collection stage by developing a new collection techniqueusing gelatin. We also examine the possibility of using a frozen section andstaining method to determine the age on the basis of histology of common
minke whales.
MATERIALS AND METHODS
Surveys
Earplugs collected from common minke whales were sampled off Sanriku(Pacific coast of northern Japan) and Kushiro (Pacific coast of eastern
Hokkaido) in Japan during 2007–2009. The samples were collected by theJapanese Whale Research Programme under a Special Permit in the Western
North Pacific - Phase II (JARPN II) Coastal Components off Sanriku andKushiro (Bando et al. 2008, Kishiro et al. 2008, 2010, Yasunaga et al. 2009,2010, Yoshida et al. 2009). This study was approved by the Japanese
government in compliance with Article VIII of the International Convention
for the Regulation of Whaling. The research operations were headed by theInstitute of Cetacean Research and the National Research Institute of Far
Seas Fisheries (Government of Japan 2004). Common minke whales for thisstudy were collected off Sanriku and Kushiro during spring (April–May)and autumn (September–October), when the maximum limit of 60
individuals is permitted. The survey area was within 50 nautical miles fromthe ports of Ayukawa and Kushiro. Four small whale catching boats (30.0–
47.3 GT) were employed as sampling vessels. All captured whales werelanded at the land station of each port for biological examination.
Earplug collection using gelatin
A total of 174 trials on the common minke whale were conducted throughthe platform of JARPN II programmes (coastal component) during 2007–2009 (Table 1). We used some biological datasets, including body length,sex, weight of testis, and number of ovulations. Based on Kishiro et al.
(2010) and Yoshida et al. (2009), a male with a testicle weighing more than
290 g and a female with at least 1 corpus luteum or albicans in the ovaries
were regarded as sexually mature.

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Table 1. Number of earplugs examined in the present survey by the
programme in each year.

Year

Research programme

2007

JARPN II

2008

JARPN II

2008

JARPN II2009

JARPN II2009

JARPN II

Coastal component off
KushiroCoastal component off
Sanriku
Coastal component off
KushiroCoastal component off
Sanriku
Coastal component off
KushiroNumber ofindividuals
38
47
20
40
29

Left earplugs were collected by using gelatin, and right earplugs werecollected by using a standard procedure. Gelatin was selected as theembedding agent because it has good adhesiveness to the earplug, it is easy
to handle in the field, and it is transparent, safe and non-toxic. The standard
collection procedure was to cut open the external auditory meatus and pickout the earplug directly using tweezers. During the collection using gelatin,
the external auditory meatus was impregnated with 40% liquid gelatin in
prefilled syringes after opening the corium of the dermis, followed bycooling of the gelatin with coolant gas. Both earplugs were preserved in
10% neutral buffered formalin solution. In the laboratory, the earplugs were
cut flat along the central axis of the core using a sharp blade according to
Lockyer (1972). Then, the earplugs were ground on a wet stone to expose
the NL and the growth layers and to smooth the surface. The cut surfaces ofthe earplugs were then examined under water using a stereomicroscope(Olympus SZX10, magnification: 3.2-31.5). Age was determined by reading
the growth layers appearing on the bisected surface of the earplug, assuming
annual deposition of growth layers (a pair of dark and light laminae
accumulated per year; Maeda 2012). In case the age could not be
determined, we categorized it according to one of three reasons, such as
obscure formation, fracture of the core, and lost NL. Photographs of all
earplugs were taken using a digital camera attached to the stereomicroscope(Nikon D90), and the entire core length along the central axis and earplug
length were measured using an image analysis software (ImageJ, a publicdomain, Java-based image processing programme, National Institutes of
Health, USA; Fig. 2).

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Frozen sectioning and staining
The normally prepared earplug sections that had clear growth layers wereselected for frozen sectioning. Frozen sections were prepared according to
the method described by Kawamoto (2003) using a Multi-purpose
Cryosection preparation kit (SECTION-LAB Co. Ltd., Japan). First, theearplugs were rapidly frozen in cooled hexane coolant. Then, the frozen
earplugs were placed in a container filled with embedding gel and frozen
completely in cooled hexane. The frozen block was then removed from thecontainer, fixed to the sample stage, and attached to a cryomicrotome (Leica,
CM3050S). The blocks were sectioned at 4–10 µm. The cutting surface wasrotated by 90° from the normal surface which is the normally prepared
earplug section. Then, the sections were stained in separate trials withtoluidine blue (10 s), hematoxylin (5 min) and eosin (15 s), Sudan III (30–
60 min), Sudan VII (30–60 min), and alizarin red S (5–10 min), eachstaining method being used separately and not sequentially on each section.
Stained sections were mounted with mounting medium (SCMM-R2;
SECTION-LAB Co. Ltd., Japan). Photographs of the stained sections weretaken by a digital camera attached to a light microscope. We also analysed
the gray value (8 bits / pixel) from the photographs by using image analysis
software Image J. Elements in the section were analysed with an energy
dispersive X-ray spectrometer (EDS).
RESULTS
Earplug collection using gelatin
Readability was improved in the samples collected using gelatin for each
body length class of whale sampled (Fig. 3). Particularly, in animals withless than 7 m body length class, NL loss was reduced in the earplugs
collected using gelatin. This clearly indicated that embedding earplugs ingelatin prevented breakage and loss of the NL (Fisher’s exact test, P < 0.05).
Figure 4 illustrates an example of a pair of earplugs. An earplug from theright external auditory meatus collected by the standard procedure had lost
the NL; whereas, the gelatinized earplug from the other side held the NL.
One year difference was found in age counting. As just described, age
readability was improved in the earplugs collected using gelatin.
Figure 5. shows the change in the proportion of animals by age class forwhich age readability was improved by earplug collection using gelatin. The
results showed that earplug collection using gelatin was effective in the <12-year-old whales and particularly in the 1–3 year age class (CochranArmitage test, P < 0.05).

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Fig. 3. Comparison between proportions (%) of readable earplugs by thestate of preservation. none: without gelatin embedding, embed.: with gelatin
embeddingFig. 4. Comparison of age readings from earplugs of a single animal (a)without gelatin embedding and (b) with gelatin embedding. Dotted linedelimits the gelatinised area. The ruler at the center is graduated inmillimeters. NL: neonatal line.

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The mean total earplug length increased with age (6.19 mm in 1–3-year-old
whales, 24.19 mm in more than 20-year-old whales; Fig. 6). The mean
length of the outer covering (difference between total earplug length andcore length) increased with age in each age class (Jonckheere-Terpstratrent
test, P < 0.05).
Frozen sectioning and staining
Although fat cells were stained, growth layers could not be identified infrozen sections (4 μm) stained with Sudan III and Sudan VII. Hematoxylin
and eosin, and toluidine blue also provided very poor results. However,alizarin red S was the most useful stain in identifying the growth layers(Table 2). Thick stained layers were observed in the core stained withalizarin red S (Fig. 7). After analyzing the gray value from the photographsof earplugs stained with alizarin red S, thick stained layers were observed
periodically in the core (Fig. 8). It is known that while interlayer space
between growth layers is wide and irregular in the immature stage, it
becomes narrower in the mature stage (Lockyer, 1984b). Since it was close
to the NL in the photographs as well, any sites considered to be in theabove-mentioned characteristic of growth layers. As a result, thick stained
peaks were periodically observed, the number of which was determined to
be fourteen that corresponded to the numbers counted under thestereomicroscope. Most probably, these alizarin red S-stained areas reflected
the growth layers. Alizarin red S solution is known to detect calcium. We
analysed a part of the well-stained area by EDS in order to determine thepresence of calcium. Except for the gold used for pre-treatment, calcium
was the only dominant component (Fig. 9), revealing that the stained area
had calcium.

Fig. 5. Changes in the proportion of animals whose age readability
was successfully improved with the use of gelatin embedding with age
class. The bar represents the number of individuals in which growthlayers existed in both earplugs in each age class.

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Fig. 6. Growth of the earplug in relation to age. a) Changes in the gaps oflength between the core length and total length of the earplug and its
standard deviation in relation to age class. b) Changes in the length of
earplugs and the earplug core in relation to age class. Bars represent
standard deviation in each age class.

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Fig. 7. The core surface of the growth layers is inside the dotted line.
Arrowheads show the thickly stained laminae.

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Fig. 8. The graph shows dark and light colouring on the dotted line in the lower photograph.

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Table 2. Assessment of staining conditions for each staining solution.

Staining solutions

Assessment

Toluidine blue

Poor

Hematoxylin & eosin

Poor

Sudan III

Moderate

Sudan VII

Moderate

Alizarin red S

Excellent

DISCUSSION
Earplug collection using gelatin
Total earplug length and the difference between earplug and core length also
increased with age. It was considered that the outer covering that covers thecore was not present in younger earplugs, but it became thicker with age,which would stabilise the earplug. Since the NL, which is important for age
estimation, is formed at the apical portion of the core or the border between
the outer covering and the core, an earplug with an immature outer covering
could break and the NL could be lost. Such cases tended to occur inindividuals of less than 7 m body length during the standard collection
procedure. No NLs were lost, and the fracture on the core was reduced
when the earplugs were collected using gelatin. These results suggested thatcollecting earplugs using gelatin could be effective for individuals of lessthan 7 m body length and an immature outer core covering. Amongindividuals that had the entire suite of growth layers in both right and left
earplugs, the proportion in which age could only be determined using theearplugs embedded in gelatin was highest in whales aged 1–3 years, which
decreased until it was zero after the age of 12 years. Therefore, thecollection method in which gelatin was used as the embedding agent was
the most effective for the 1–3-year age class and less effective after the ageof 12 years. As a result, collecting earplugs using gelatin would enable theage estimation rate to improve much more in young and small individuals
that have soft earplugs with underdeveloped outer coverings. In contrast, we
did not recognise much improvement in the age estimation rate for thoseindividuals in the greater than 7 m body length class and those aged above
12 years. The outer covering was well developed, and the earplug itself wasstabilised in these individuals; thus, the collection could be performed by
the existing method without any loss or breakage of the NL. The decision touse gelatin for collecting earplugs should depend on the body length (< 7 m)
and the earplug condition at the time of dissection of the external auditory
meatus. The collection technique using gelatin can be employed in

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accordance with the shape and hardness of earplugs for other species.
However, each species has variously shaped and sized earplugs; therefore, itwill be necessary to examine the specific features of other species earplugs
in order to apply the collection method using gelatin.

Fig. 9. The graph shows the analysis of a part of a well-stained area
using energy dispersive X-ray spectrometry. Except for the gold used
for pre-treatment, calcium was the only dominant component.

Frozen sectioning and staining
We found that alizarin red S was the best staining solution for identifying
growth layers. Even with the use of image analysis software, it produced thesame results as those of the standard method of age estimation. EDS
analysis showed that calcium was contained in the dyed portion. It was also
found that calcium accumulation was recorded as layers in the earplug andthese layers corresponded to the growth layers. Thus, it could be possiblethat we can get further information from calcium content in earplugs. A pastchemical analysis study using fin whale earplugs indicated that calcium
tended to collect less in the dark layer and more in the light layer (Hamada
et al. 1989). Since a relationship between formation of the growth layer andchange in the calcium density has been suggested, the layers that dyed
darker with alizarin red S reflected the lighter layer or a layer formed during
the feeding period. Earlier age estimation studies focused on fat content inlight layers, such as direct counting by the naked eye or Sudan staining;
however, there is potential for improving the readability of unclear growthlayers while using calcium. As this was a preliminary study, it will benecessary to further clarify the relationship between formation of the growthlayer and change in the calcium density while increasing the number of

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samples and directly assessing the calcium content along the growth layers
using wavelength dispersive X-ray spectrometry.
In past studies, the techniques of X-ray photography (Masaki 1968, Lockyer
1974), bleaching the earplug with hydrogen peroxide (Lockyer 1974) andusing an image processing system (Kato et al. 1988) were developed to
improve the readability of earplugs in fin whales, sei whales (Balaenoptera
borealis), and Antarctic minke whales (Balaenoptera bonaerensis).
Unfortunately, these methods have not led to any practical use. However,
the earplugs of common minke whales remain a valid age tool. Particularly,
the methods presented in this paper to improve the earplug readings could
make a significant contribution for calibrating the racemisation method forcommon minke whales. Therefore, it will be necessary to continue toimprove the readability as much as possible from the collection stage to theage estimation stage.
ACKNOWLEDGMENTS
We thank all the survey leaders of JARPN II; Mutsuo Goto, Takeharu
Bando, Genta Yasunaga (Institute of Cetacean Research), Toshiya Kishiro,and Hideyoshi Yoshida (National Research Institute of Far Seas Fisheries)
for supporting this study. We also thank the crew of the research vessels andstaff of the research station. Finally, we are indebted to Yoshihiro Fujise(Director of the Institute of Cetacean Research) for his valuable support.
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NAMMCO Scientific Publications, Volume 10