SC/F14/J12

Preliminary estimation of the age of Antarctic minke
whales based on aspartic acid racemization
GENTA YASUNAGA, TAKEHARU BANDO AND YOSHIHIRO FUJISEThe Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
Contact e-mail:yasunaga@cetacean.jp

ABSTRACTTo examine the approach of age estimation in Antarctic minke whales by ratio of aspartic acidenantiomers, the eye lenses of 18 whales and of 20 fetuses were measured. In the age estimation
equation, the specific coefficient of hydrolysis effect in preparation and the constant conversionof aspartic enantiomers (Kasp) were estimated. We found that the D/L ratio of aspartic acid in
the lenses of minke whales had increased by 0.0102 in the hydrolysis process (7 hr at 108°C),
and estimated that the Kasp was 1.96×10-3 ( /year) calculated from the earplug age and the age
index with the D/L ratio of aspartic acid in minke whales. Consequently, formulae Age (year) =511×Ln ((1+D/L)/(1-D/L)) -13.2 was obtained.
KEYWORDS: ANTARCTIC MINKE WHALE; AGE ESTIMATION; RACEMIZATION;ASPARTIC ACIDINTRODUCTION
Age composition is one of the life history parameters needed to assess the dynamics of whale populations.
In the past, criteria such as body length, growth layers of the earplug (Purves, 1955; Zenitani and Kato,
2006), teeth (Scheffer and Myrick, 1980) and tympanic bulla (Christensen, 1995), and color of eye lenses(Nishiwaki, 1950), have been used to estimate physiological age in many species. For baleen whales,
growth layers deposited in the earplug are the most useful criterion because they indicate chronologicalage. Earplug-age determination has become standard procedure in stock assessment and management
decisions for baleen whales. Earplug-age determination has advantages in time- and cost-efficiencies andexisting abundant historical data; however, there is age estimation bias among proficient readers andreadable earplug whale species. Some species and individuals don’t have readable earplugs. Therefore,chemical age estimation is expected to gain support in the field as a reader is unnecessary.
The principles of age estimation with aspartic acid racemization had been elaborated on throughcomparison of the ratio of aspartic acid enantiomers and counting the number of growth layers in the teethof tooth whales and pinnipeds study (Bade et al., 1980; Bada, 1984). This method yielded the simple
kinetic equation for the racemization reaction and the constant conversion of aspartic enantiomers (Kasp)
which have to be determined through experiments used with animals which have available age data, andthe hydrolysis effect should be further considered.
Nerini (1983) revealed the clear relationship between the earplug ages of fin whales which are easily
determined by this method and their D/L ratios of aspartic acid in their eye lenses, whereas he ignored thehydrolysis effect. George et al. (1999) obtained the age estimation equation with aspartic enantiomers ofbowhead whales from Alaska, whereas the Kasp were extrapolated by the previous studies of fin whales
and humans. Auðunsson et al. (2013) obtained the age estimation equation with aspartic enantiomers ofcommon minke whales from the North Atlantic Ocean, whereas the Kasp were extrapolated from thesample of Antarctic minke whales. Thus, certain age data, fetus samples and various biological data areneeded to determine the specific values in the age estimation equation with aspartic enantiomers of baleen
whales. In Antarctic minke whales taken from JAPRAII surveys, conclusive age data, fetus samples,
reproductive status and other biological data are available for these estimations.
The objective of this report is to obtain the age estimation equation with aspartic acid enantiomers in thelenses of Antarctic minke whales. Then, we estimate the Kasp and correct for the effect of hydrolysis
using the lens sample from Antarctic minke whales and their fetuses from the 2005/2006 and 2007/2008

1

JAPRAII surveys.
MATERIALS AND METHODS
Materials
Antarctic minke whales were collected from Area IV (70º-130ºE, Figure 1) in the 2005/2006 and2007/2008 JAPRAII surveys. In the field, left eye lens samples of 18 females and 20 fetuses were storedin polyethylene bags at -80°C until analysis.
Biological information
The weight of the fetuses was measured by the marine scale (M11000, Marel Co. Ltd.). To convert the
fetal weight (g) into fetal age (t: day) we used the equation of Kato and Miyashita (1991):
t=W1/3/0.24+74(1)

Table 1 shows the number of fetus samples used in the chemical analysis divided by sex, body weight andfetus age (day). The whale body length was measured from the jaw to the tail notch by researchers
onboard the vessels. Ages of the whales were determined by counting the growth layers on the core of theearplugs (Purves, 1955; Zenitani and Kato, 2006).
Table 2 shows the number of whale samples used in the chemical analysis divided by sex, body lengthand earplug ages (year).
Preparation of samples
The lens samples were stripped externally to avoid contamination and homogenized with Tris-buffer(200mM Tris, 150mM NaCl, pH 8.0) using ultrasonic disruptors. The homogenate was centrifuged at
1,5000 × g for 15 min. at 4°C, and it was then desalted with acetone and air-dried. The sample redissolved in water was then set up within a glass tube for hydrolysis and freeze-dried. The purified
samples were lyophilized in tubes and were hydrolyzed in the gas-phase 6N-HCl for 7 hr at 108°C (PicoTag Work Stations, Waters, Tokyo). The hydrolysates were evaporated under reduced pressure.
For validation of the effect on hydrolysis, the purified fetus samples, 07/08-AM533F, were hydrolyzed in
the gas-phase 6N-HCl for 7, 14, 21, 28 and 35 h at 108°C, respectively.
Apparatus and quantitative analysis
The hydrolysates were dissolved and incubated in 0.1N-HCl and Borate buffer (0.1M, pH10.4), and wereincubated with o-phthalaldehyde (OPA) and n-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) to form
diastereoisomers. The D/L ratio of aspartic acid was determined using RP-HPCL (Alliance ® HPLC
systems e2696, Waters) with a Nova-Pak ODS column (3.9mm ×300mm, Waters) using fluorescence
detection (344 nm excitation wavelength and 433 emission wave length). Elution was carried out with asimple isocratic adsorption of 3% acetonitril+3% tetrahydrofuran /0.1M acetate buffer pH6.0 in 45 min at
a flow rate of 0.8 ml/min, at 23°C. Sensitivity of intensity for both enantiomers of aspartic acid wascorrected by 100ppm DL-Aspartic acid (minimum 99% TLC, SIGMA-ALDRICH) solution.
The precision and accuracy and linearity of the D/L ratio were assessed with the 3 mixtures of D- and Laspartic acid standards, which were 1.5ppm: 100ppm, 3.0ppm: 100ppm, 5.0ppm. Good reproducibility
and linearity in the D/L ratio range from 1.5 and 5.0 were obtained (Table 3).
Method of age estimation
To estimate ages by ratio of enantiomers of aspartic acid, we used the formula of Bada et al. (1980):
2Kasp・t =Ln((1+D/L)/(1-D/L))-Ln(1+D/L) /(1-D/L))t=0(2)where Kasp is the rate constant of conversion between L and D enantiomers, t is the age of the whale,
D/L is the ratio of the enantiomers in the lens, and “t=0” is the ratio at birth.
Statistics
The correlations between ratio of aspartic acid enantiomers, and hydrolysis time and fetal age wereassessed by the Pearson correlation test (Zar, 1999). The Kasp and the constant terms at birth wereassessed by linear regression analysis (Zar, 1999). To estimate for the error of the predicted value of theconstant term at birth, we used the equation of Devore and Berk (2012):2

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SE(age)=δ2 ቂ +݊(‫ ∗ݔ‬−‫) ̅ݔ‬2
ܵ‫ݔݔ‬

ቃ

(3)
where x* is the predicted x value, SE(age) is the predicted value used by the regression equation, n is thenumber of x data, Sxx is the variance of x×n, and δ is the standard error in the regression equation. A pvalue of less than 0.05 was used as a criterion on statistical. These statistical analyses were executed by
PASW Statics 17.0 for Windows (SPSS Co. Ltd.).
RESULTS
Effect for the acid hydrolysis
Examining the racemization effect on hydrolysis intensity, the purified lens samples of fetal Antarctic
minke whales, 07/08-AM533F, were hydrolysed several times. The D/L ratios of the samples hydrolyzed
for 7, 14, 21, 28 and 35 h at 108°C were 0.0285, 0.0396, 0.0486, 0.0594 and 0.0694, respectively (Fig. 2).
The D/Lasp ratio correlated with fetal age (Pearson correlation test; r = 1.000, p < 0.001). The data
yielded the following linear regression equation:
Hydrolysis time (hr) = 689×D/Lasp-12.8(p<0.001, r2=0.999)(4)

Aspartic D/L ratio in whales
Table 1 shows the ratio of D/Lasp ratios in the lenses of fetuses of Antarctic minke whales, and figure 3
shows the relationships between the D/Lasp ratios and fetal ages (day). The D/Lasp ratio correlated withfetal ages (Pearson correlation test; r = 0.628, P < 0.01). The data yielded the following linear regression
equation:
Fetal age (day) = 39400×D/Lasp-768(p<0.01, r2=0.394)(5)

Table 4 shows the ratio of D/Lasp ratios in the lenses of Antarctic minke whales.

DISCUSSION
Effect for the acid hydrolysis
Bada (1984) suggested that the age estimation equation with aspartic acid enantiomers is needed toaccount for the fact that the initial D/L ratio in the system under investigation is not exactly 0 mainly
because some slight racemization takes place during the protein hydrolysis step. We compared this withthe measurement of the D/L ratio affected by the hydrolysis step and the estimation of the D/L ratio atbirth by the D/L ratio in the lenses of fetuses and their fetal ages. The correlation coefficient of the D/Lratios and the hydrolysis times were obviously higher than that of the D/L ratios and fetal ages; therefore,
the age estimation equation in our system is corrected by the hydrolysis bias. The initial D/L ratio in thelenses of whales was 0.0102 calculated in equation (5). After this, 0.0102, the hydrolysis effect, wassubtracted from the crude D/Lasp ratios in whales.
Determination of the Kasp
Table 4 shows the ratio of D/Lasp ratios in the lenses of fetuses of Antarctic minke whales corrected by
the hydrolysis effect and the age index which is “Ln ((1+D/L)/(1-D/L))”. Fig. 4 shows the relationships
between the age index in Antarctic minke whales and their earplug ages.
The single outlier at 40 years old for which Cook’s distance exceeded 2 was eliminated in the first timeregression analysis. Then, the age estimation equation is given to:Age (year) = 511×Ln ((1+D/L)/(1-D/L)) -13.2∴ 2Kasp=1.96×10-3 ( /year)(r2=0.923)(6)

Estimated age used in equation (6) and their standard deviation used in equation (3) in the aspartic acidenantiomers of the lenses of minke whales are shown in Table 4, and the relationships between theestimation age and the earplug age are shown in fig. 4.
The value of 2Kasp in this study is lower than that which was found for fin whales (2.21×10-3 in Nerini,1983), Antarctic minke whales (2.94×10-3 in Auðunsson et al., 2013) and humans (2.50×10-3 in Masters
et al., 1997). The values were inconsistent among the previous studies and this study. Bada (1984)
supported that the 2Kasp is mainly affected by the temperature of the lens during its life-span and thehydrolysis process during preparation, and recommended that the hydrolysis effect be corrected by the Ln3

((1+D/L)/(1-D/L)) at birth. We suggest direct correction of the hydrolysis effect in the system, because
estimating the hydrolysis effect is more precise while estimating the age index value at birth is difficult.
CONCLUSION
The present study reported the preliminary age estimation of aspartic acid enantiomers in the lenses ofAntarctic minke whales. We proposed a new approach to correct for the bias of the hydrolysis step during
preparation. In the future, this estimation method needs to be extended to some whales and individuals
which don’t have readable earplugs.

ACKNOWLEDGMENTS
We would like to thank L.A. Pastene of the Institute of Cetacean Research for helpful suggestions andcriticism of the manuscript. N. Fujii of the Research Reactor Institute of Kyoto University has been
incredibly tolerant and supportive in the racemization analysis.
REFERENCES
Auðunsson, G.A., Nielsen, N.H., Víkingsson, G.A., Halldórsson, S.D., Gunnlaugsson, þ., Elvarsson, B.þ.,Kato, H. and Hansen, S.H. 2013. Age estimation of common minke whales (Balaenoptera
acutorostrata) in Icelandic waters by aspartic acid racemization (AAR),-AAR and earplug readings ofAntarctic minke whales (B. bonaerensis) used as a reference. Paper SC/F13/SP15 presented the
Icelandic a review panel of experts in a workshop, Reykjavik, February 2013 (unpublished). 17pp.
Bada, J.L., Brown, S. and Masters, P.M. 1980. Age determination of marine mammals based on aspartic
acid racemization in the teeth and lens nucleus. Rep. Int. Whal. Commn. Spec. (Special Issue 3), 113-8.
Bada, J.L. 1984. In vivo racemization in mammalian proteins. Methods Enzymol. 106:98-115.
Christensen, I. 1995. Interpretation of growth layers in periosteal zone of tympanic bulla from minke
whales Balaenoptera acutorostrata. In A.S. Brix, L. Walløe and Ø. Ulltang (eds.). Whales, seals fishand man. Elsevier Science, Amsterdam, pp413-23.
Devore, J.L. and Berk, K.N. 2012. Modern mathematical statistics with applications second edition,
Springer, New York, NY, 845pp.
George, J.C., Bada, J., Zeh, H., Scott, L., Brown, S.E., O'Hara, T. and Suydam, R. 1999. Age and growthestimates of bowhead whales (Balaena mysticetus) via aspartic acid racemization. Can. J. Zool. 77:57180.
Kato, H. and Miyashita, T. 1991. Migration strategy of southern minke whales in relation to reproductive
cycle estimated from foetal lengths. Rep. Int. Whal. Commn. 41:363-69.
Masters, P.M., Bada, J.L. and Zigler J.S. Jr. 1977. Aspartic acid racemization in the human lens during
ageing and in cataract formation. Nature 268:71-3.
Nerini, M.K. 1983. Age determination of fin whales (Balaenoptera physalus) based upon aspartic acidracemization in the lens nucleus. Rep. Int. Whaling Comm. 34:447-8.
Nishiwaki, M. 1950. Determination of the age of Antarctic blue and fin whales by the colour changes incrystalline lens. Sci. Rep. Whal. Res. Inst. 4:115-61.
Purves, P.E. 1955. The wax plug in the external auditory meatus of the Mysticeti. Discoverty Rep., 27:
293-302.
Scheffer, V.B. and Myrick, A.C. Jr. 1980. A review of studies to 1970 of growth layers in the teeth ofmarine mammals. Rep. Int. Whal. Commn. (Special Issue 3), 51-64.
Zar, J.H. 1999. Biostatistical analysis, 4th ed., Prentice Hall, New Jersey, USA, 663pp.

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Zenitani, R. and Kato, H. 2006. Temporal trend of age at sexual maturity of Antarctic minke whales
based on transition phase in earplugs obtained under JARPA surveys from 1987/88-2004/05. In: Reportof the intersessional workshop to review data and results from special permit research on Minke Whalesin the Antarctic. Tokyo. 4-8 December 2006. SC/D06/J15. 9pp.

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Table 1. Biological information and ratios of aspartic enantiomers of fetuses of Antarctic minke whales
Ratio of aspartic
Sample No.
Sex
Body weight (kg)Foetal age (day)enantiomers (D/L)
07/08-AM363F
M
49.4
227
0.025407/08-AM380F

M

19.0

185

0.024807/08-AM383F

F

64.1

241

0.026007/08-AM392F

M

29.9

203

0.025607/08-AM393F

M

19.2

186

0.024007/08-AM461F

M

59.3

236

0.025407/08-AM474F

M

35.8

211

0.024607/08-AM475F

F

63.2

240

0.025207/08-AM481F

M

21.9

191

0.025507/08-AM502F

M

78.5

252

0.025407/08-AM504F

M

14.0

174

0.024307/08-AM516F

F

22.1

191

0.025407/08-AM523F

M

108.0

272

0.025207/08-AM527F

M

34.6

210

0.025407/08-AM531F

M

100.0

267

0.025507/08-AM533F

F

194.5

315

0.026407/08-AM542F

F

98.0

266

0.025607/08-AM550F

F

58.8

236

0.024805/06-AM588F

M

49.0

226

0.025505/06-AM820F

F

53.6

231

0.0259

Table 2. Biological information of Antarctic minke whales
Sample No.
Sex
Body length (m)Earplug age (year)

05/06-AM348

F

9.56

14

05/06-AM349

F

9.28

21

05/06-AM352

F

8.64

9

05/06-AM361

F

8.84

26

05/06-AM372

F

9.02

40

05/06-AM382

F

7.82

7

05/06-AM386

F

7.84

8

05/06-AM398

F

7.96

7

05/06-AM488

F

6.61

4

05/06-AM498

F

7.14

4

05/06-AM517

F

5.82

3

05/06-AM539

F

5.31

1

05/06-AM565

F

5.44

2

05/06-AM592

F

7.43

4

05/06-AM603

F

7.23

6

05/06-AM615

F

7.03

7

05/06-AM630

F

7.32

5

05/06-AM634

F

6.21

3

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Table 3. Precision of the D/L ratio of mixtures of D- and L- aspartic acid standards

Reagentn

Asp-L 100ppm D1.5ppm
Asp-L 100ppm D3.0ppm
Asp-L 100ppm D5.0ppm

10
10
9

Age index*
SD
0.02
0.02
0.05

Average
1.43
2.99
4.75

error (%)
1.3
0.8
1.1

Table 4. The ratio of aspartic acid enantiomers, age index and estimation age of Antarctic minke whales
Sample No.

Ratio of aspartic enantiomers (D/L)
Actual value

Correction value*

05/06-AM348

0.03330.0231

05/06-AM349

0.041605/06-AM352

0.033305/06-AM361
05/06-AM372

Age index**

Estimation age (year)
AgeSE

0.046310.4

2.0

0.03140.062918.9

1.9

0.0231

0.046210.4

2.0

0.04980.03960.079327.3

1.7

0.04960.03940.078905/06-AM382

0.02950.01930.03856.5

2.1

05/06-AM386

0.03070.02050.04117.8

2.1

05/06-AM398

0.02870.01850.03715.8

2.1

05/06-AM488

0.02650.01630.03263.5

2.1

05/06-AM498

0.02770.01750.03504.7

2.1

05/06-AM517

0.02410.01390.02791.0

2.2

05/06-AM539

0.02560.01540.03082.5

2.2

05/06-AM565

0.02950.01930.03866.5

2.1

05/06-AM592

0.02910.01890.03786.1

2.1

05/06-AM603

0.02940.01920.03856.5

2.1

05/06-AM615

0.02890.01870.03755.9

2.1

05/06-AM630

0.02780.01760.03524.8

2.1

05/06-AM634

0.02570.01550.03092.6

2.2* (hydrolysis effect correction value): Actual value-0.102** (Age index by Bada 1980): LN(1+D/L)/(1-(D/L))

7

Sampling area

Figure 1. Sampling area of Antarctic minke whales in 2005/2006 and 2007/2008 JAPRAII researchcruisesAcid hydrolysis time (hour)

40

30

20

10

0
0.00000.02000.04000.06000.0800

Ratio of aspartic enantiomers (D/L)

Figure 2. Relationship between acid hydrolysis times and ratio of aspartic enantiomers (D/L) in lenses offetuses of Antarctic minke whales

8

Ratio of aspartic enantiomers (D/L)

350
300
250
200
150
100
0.02250.0250Fetal age (day)0.0275Figure 3. Relationship between fetal age and ratio of aspartic enantiomers (D/L) in fetuses of Antarctic
minke whales

Age by earplug (year)

50
40
30
20
10
0
0.00000.02500.05000.07500.1000Age index calculated by aspartic enantiomers*
Figure 4. Relationship between age index calculated by aspartic enantiomers in lenses of Antarctic minke
whales and age determined by earplug*: Age index=Ln ((1+D/L)/(1-D/L))

9

30
Earplug age (year)
10
20

30

20

0
20

30

30

10

20

0

0

10

0

10

Estimation age by aspartic acid enantiomers (year)
Figure 4. Relationship between earplug age and estimation age by aspartic acid enantiomers in lenses ofAntarctic minke whales

10