Technical Reports of the Institute of Cetacean Research (2019)

pp. 9–13

Technical Report (not peer reviewed)

Age estimation of Antarctic minke whales based on aspartic acidracemization: technical development
Genta Yasunaga*
Institute of Cetacean Research, 4–5 Toyomi-cho, Chuo-ku, Tokyo 104–0055, Japan*Contact e-mail: yasunaga@cetacean.jp

ABSTRACTAt present, examination of earplugs is the only practicable means to obtain age data at the annual scale inbaleen whales. It is the only method providing age data accurate enough for population-level analyses such as
the Statistical Catch-at-Age Analysis (SCAA) of Antarctic minke whales. Because there are a number of whales
sampled with unreadable earplugs, the feasibility of using other techniques for age determination is being
investigated by scientists at the Institute of Cetacean Research with the aim of determining the age of thosesamples where earplugs does not work. One of those techniques is based on enantiomers of aspartic acid ineye lens, the aspartic acid racemization (AAR) technique. This paper presents a brief review of the technicaldevelopment of the AAR technique at the Institute of Cetacean Research, and the preliminary results of using
this technique for age determination of Antarctic minke whales.

INTRODUCTION
Age is one of the most important life history parameters
for assessment and management of marine living resources. In baleen whales, age has been determined using
a variety of methods such as examination of baleen plates(Nishiwaki, 1951; Zenitani and Kato, 2010), earplugs(Lockyer, 1984) and tympanic bulla (Christensen, 1995).
Counting of the growth layers deposited in the earplugs isthe most accepted technique for determining chronological age of baleen whales (Lockyer, 1984). Earplug-basedage determination has the advantages that it is time- andcost-eﬃcient, and the technique can be used on available historical samples; however, unreadable growthlayers form in some individuals baleen whales (Maedaet al., 2013; George et al., 1999). In such cases alternative
methods of age determination are required.
Indices of chronological age of whales have beendeveloped using molecular approaches. For example
length of telomeres, nucleoprotein caps ﬂanking DNA(Olsen et al., 2012), and methylation levels at the C5
position of cytosine residues adjacent to guanidine
residues (Polanowski et al., 2014) were examined in relation to age using skin of humpback whales (Megapteranovaeangliae) in the Gulf of Maine. The feasibility of theDNA methylation technique to determine age in Antarctic
minke whales (Balaenoptera bonaerensis) has been evaluated by scientists at the Institute of Cetacean Research(ICR) (Goto and Inoue, 2018). Currently, these epigenetic
approaches face challenges such as measurement of indices error and whale speciﬁc biological processes, whichmakes it diﬃcult to interpret the age of whales.
Regarding biochemical approaches, the Advanced Glycation End (AGE) products was proposed as one of thepossible factors underlying the functioning of the biological clock for mammals (Severin et al., 2013), however norelationship was observed between AGE levels and age
in bowhead whales (Rosa, 2006). Another technique isbased on enantiomers of aspartic acid in eye lens, the aspartic acid racemization (AAR) technique, which is being
developed at the ICR.
This paper presents a brief review of the technicaldevelopment of the AAR technique at the ICR, and shows
preliminary results of using this technique for age determination of Antarctic minke whale.

PRINCIPLE OF THE AGE ESTIMATION BY THE
ASPARTIC ACID RACEMIZATION TECHNIQUE
The AAR technique for determining age is based on
temporal changes of the ratio of D and L-enantiomers
of aspartic acid in mammals (Helfman and Bada, 1975)(Figure 1). The technique is based on the principle that
D-aspartic acid accumulates logarithmically with age, andthe crystalline in the core of the lens has been conserved
chemically since it is formed at the fetus stage and ismetabolically inactive for all of the animalʼs life (Masters9Technical Reports of the Institute of Cetacean Research (2019)

Figure 1.

Figure 2.

Scheme of racemization of aspartic acid.

Research area of JARPAII in the 2005/2006 and 2007/2008 austral summer seasons.

et al., 1977).
Masters et al. (1977) and Bada et al. (1980) proposedthat the racemization of amino acids follows a ﬁrst-orderreversible rate law, where the racemization equation is:

2kAsp ⋅ t = Log e [ (1+ D /L) / (1 − D /L) ]{}–Ln [1+( D /L)0 ] [1 − ( D /L)0 ] .

(1)A linear regression model is constructed using this expression, where D/L is the ratio of D- and L-aspartic acids,t is any given time during racemization, and the logarithmic term at t=0 describes the amount of D-aspartic acidformed at birth.

RESEARCH ON ASPARTIC ACID RACEMIZATION AT
THE INSTITUTE OF CETACEAN RESEARCHAt the ICR, the feasibility of using the AAR technique todetermine the age of Antarctic minke whales has been
investigated (see details in Yasunaga et al., 2017). The
JARPA and JARPAII research programs in the Antarctic
have provided suﬃcient biological information on thisspecies that makes assumptions and extrapolations ofkey parameters used in this technique unnecessary. For
example kAsp and (D/L)0 in Eq. (1) can be determined by direct comparison with earplug-based estimated ages, and(D/L)0 can be estimated using available foetal samples.
10Samples
Antarctic minke whales used in this study were sampled
in the Indo region of the Antarctic, which corresponds
to the International Whaling Commissionʼs (IWC)ʼs management Area IV (70°–130°E), south of 62°S (Figure 2).
Whales were sampled during the austral summer seasons2005/2006 and 2007/2008 by JARPAII surveys.
At the ﬁeld, scientists collected the left eyeball from
20 foetuses and lens samples from 18 female Antarctic
minke whales. The samples were stored in polyethylenebags at −80°C until analysis.
Preparation of lens of Antarctic minke whale
Lenses of all samples (foetuses and females) were rinsed
ﬁrst with phosphate-buﬀered saline (Figure 3). Then
their outermost layers were removed with mucus, andthe cores were dissected out using a surgical knife. Approximately 10 mg of core samples were homogenizedwith 1 ml of tris-buﬀer (200mM Tris, 150mM NaCl, pH8.0) using an ultrasonic homogenizer. The homogenate
was centrifuged at 15,000×g for 15 min at 4°C, and 100 µlof the supernatant was then desalted with acetone andair-dried. They were hydrolyzed in the gas-phase HCl(6N-HCl) for 7 h at 108°C (Pico Tag Work Stations, Waters,Tokyo) (Figure 4). The hydrolysates were evaporated

Age estimation of Antarctic minke whales based on aspartic acid racemization

Figure 3.

Lens of Antarctic minke whale rinsed with phosphate buﬀer.

Figure 5.

Figure 4.

Hydrolysis of lens of Antarctic minke whale using
Pico Tag Work Stations (Waters, Tokyo).

under reduced pressure.
Laboratory procedures
The Asp D/L was determined using HPCL (Alliance®
HPLC systems e2696, Waters) with a Nova-Pak ODScolumn (3.9 mm×300 mm, Waters) using ﬂuorescence
detection (344 nm excitation wavelength and 443 nm
emission wave length) (Figure 5). Elution was carriedout with an isocratic adsorption of 3% acetonitrile +3%
tetrahydrofuran/0.1M acetate buﬀer pH 6.0 at a ﬂow rateof 0.8 ml/min and column temperature of 23°C. Then,
70 µl of borate buﬀer (0.1M, pH 10.4), 5 µl of n-tertbutyloxycarbonyl-l-cysteine and 5 µl of o-phthalaldehyde
were successively added to 5 µl of the hydrolysate dissolved in 0.1 N-HCl. The measured Asp D/L was calibrated
against real ratios in the standard solutions and then it
was corrected for hydrolysis eﬀect under our laboratory
conditions, which was represented as (D/L)act.

HPCL system (Alliance® HPLC systems e2696,Waters) used for ﬂuorescence detection.

Results
Table 1 shows (D/L)act and the age index calculated for the18 female Antarctic minke whales. The (D/L)0 which is oneof two speciﬁc coeﬃcients of AAR, was determined using
lenses of foetuses at various developing stages because
their (D/L)act can be approximated to those at birth. However, precision of the (D/L)0, which is estimated using foetuses was unsatisfactory ((D/L)0 =0.0134; SE=3.78×10−4;
95% conﬁdential interval 0.0121–0.0147).
Taken together, the slope of the age estimation equation was examined for two cases. Figure 6 shows the relationships between the age indexes and earplug ages. The
single outlier at 40-years-old for which Cookʼs distanceexceeded 2 was eliminated in the ﬁrst regression analysis.
The two equations (cases) of age estimation are shown
below as Eq. (2) and Eq. (3). In Eq. (1) linear regression
analysis were re-performed to determine kAsp substituting (D/L)0 =0.0134 using dataset of the (D/L)act and theearplug ages in the 17 whales as follows:{}

Log e [1+ ( D /L)act ] [1 − ( D /L)act ]= 1.79 ×10−3 × earplug age (year) + 0.0268,∴ p < 0.001, r 2 = 0.890, kAsp = 8.94 ×10−4 ,SE (2kAsp ) = 1.52 ×10−4 .

(2)In Eq. (3) linear regression analysis was performed todetermine kAsp and (D/L)0 in Eq. (1) using dataset of the(D/L)act and the earplug ages in the 17 whales as follows:11

Technical Reports of the Institute of Cetacean Research (2019)
Table 1
The actual D/L ratios of aspartic acid in lens and AAR ages of Antarctic minke whales.
Sample No.

Sex

Earplug Age(D/L)actLoge[(1+D/L)/(1−D/L)]

05/06-AM348
05/06-AM349
05/06-AM35205/06-AM361
05/06-AM37205/06-AM38205/06-AM386
05/06-AM39805/06-AM488
05/06-AM498
05/06-AM517
05/06-AM539
05/06-AM565
05/06-AM592
05/06-AM603
05/06-AM615
05/06-AM630
05/06-AM634

FF
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F

1421926407874431246753

0.02230.03280.02220.04320.043
0.01730.019
0.01640.01360.01510.0106
0.01240.01740.01690.01730.01670.01520.01250.0446
0.06560.04440.08650.086
0.03470.038
0.03290.02720.03020.02120.02490.03480.03370.0346
0.03330.03050.025

Age estimated by the AAR*
AgeSE

95% CI

10.6
19.8
10.6
28.9

1.5
2.2
1.5
2.9

8.1–14.2
16.1–25.0
8.2–14.2
24.2–35.7

6.3
7.8
5.6
3.1
4.4
0.5
2.1
6.4
5.9
6.3
5.8
4.5
2.2

1.2
1.3
1.2
1
1.1
0.8
0.91.2
1.2
1.2
1.2
1.1
0.9

4.4–9.1
5.6–10.8
3.7–8.2
1.4–5.3
2.6–6.8−0.9–2.3
0.5–4.1
4.4–9.1
4.0–8.6
4.3–9.1
3.8–8.5
2.8–7.0
0.7–4.2* Ages estimated by the AAR were derived from Eq. (3)Figure 6.

Relationship between age indexes, Loge {[1+(D/L)act]/[1−(D/L)act]}, and ages of earplugs of Antarctic minke
whales: open circle was excluded from simple
linear regression analysis as an outlier, and a solidand broken line is calculated by Eq. (2) and Eq. (3),respectively.

{}

Log e [1+ (D /L)act ] [1 − ( D /L)act ]= 2.30 ×10−3 × earplug age (year) + 0.0201,
2−3∴ p < 0.001, r = 0.918, kAsp = 1.15 ×10 ,SE (2kAsp ) = 1.71×10−4 , SE (intercept) = 1.72 ×10−3 . (3)Squared correlation coeﬃcient of the Eq. (3) was
higher than that of the Eq. (2), and the SE of 2kAsp andintercept of the Eq. (3) were lower than those of the Eq.
12Figure 7.

Relationship between ages estimated by AAR andearplugs (averages and SEs estimated by bootstrap
simulation) in Antarctic minke whales.

(2). Therefore, Eq. (3) is considered more precise and accurate to estimate ages of whales than Eq. (2).
Finally, the standard errors were calculated for theages estimated by AAR. Ages estimated from Eq. (3) and(D/L)act in each whale, including their SEs and 95% conﬁdence intervals estimated by bootstrap simulation, areshown in Table 1. Figure 7 shows the relationships be-Age estimation of Antarctic minke whales based on aspartic acid racemization

tween ages estimated from earplugs, and ages (including
SEs and 95% conﬁdence intervals), estimated by the AAR.
The range of SEs of the AAR-based age estimates was 0.9
and 2.9 years, and they were within the 95% conﬁdence
interval, except for one case.

CONCLUSIONSThis study was successful in developing the AAR technique for the Antarctic minke whale. The application ofthis technique can complement the age estimation ofindividuals of this species based on earplug reading, especially for young animals with unreadable earplugs. A
few issues including bias due to cataracts require furtherconsideration in the future.

ACKNOWLEDGEMENTS
I would like to thank the captains, crews and scientists involved in the 2005/2006 and 2007/2008 JARPAII surveys.
Thanks are also due to Noriko Fujii (Research Reactor Institute, Kyoto University), for providing HCLP analysis, andTakeharu Bando (ICR) for earplug-based age data.

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