NOAA Technical Memorandum NMFS 

APRIL 1983 

ESTIMATING AGE OF SPOTTED AND SPINNER 

DOLPHINS (Stenella attenuata AND Stenella 

longirostris) FROM TEETH 

Albert C. Myrick, Jr. 

Aleta A. Hohn 

Priscilla A. Sloan 

Makoto Kimura 

Drew D. Stanley 

NOAA-TM-NMFS-SWFC-30 

U.S. DEPARTMENT OF COMMERCE 

National Oceanic and Atmospheric Administration 

National Marine Fisheries Service 

Southwest Fisheries Center 
NOAA Technical Memorandum NMFS 

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work and may be referenced in the formal scientific and technical literature. 
NOAA Technical Memorandum NMFS 

This TM series IS used for documenla!ion and timely communication of preliminary results, imterim repom. or 

special purpose inlormalion. and have not received complete formal review. editorial control, or detailed ediling 

APRIL 1983 

ESTIMATING AGE OF SPOTTED AND SPINNER 

DOLPHINS (Stenella attenuata AND Stenella 

longirostris) FROM TEETH 

Albert C. Myrick, Jr., Aleta A. Hohn, 

Priscilla A. Sloan, Makoto Kimura 

and Drew D. Stanley 

Southwest Fisheries Center 

National Marine Fisheries Service, NOAA 

8604 La Jolla Shores Drive 

La Jolla, California 92038 

NOAA-TM-NMFS-SW FC-30 

U.S. DEPARTMENT OF COMMERCE 

Malcolm Baldrige, Secretary 

National Oceanic and Atmospheric Administration 

John V. Byrne. Administrator 

National Marine Fisheries Service 

William G. Gordon, Assistant Administrator for Fisheries 
CONTENTS 

Page 

ABSTRACT ............................................................ 1 

INTRODUCTION ........................................................ 1 

DISTRIBUTIONy DEPOSITIONy AND UTILITY OF 

POSTNATAL DENTAL TISSUES ......................................... 2 

PREPARATION OF TOOTH THIN SECTIONS .................................. 3 

DEFINING DENTINAL GLGs .............................................. 6 

CEMENTUM ............................................................ 10 

SUGGESTED PROTOCOL FOR DETERMINING 

AGE FROM GLGs .................................................... 10 

ACKNOWLEDGEMENTS .................................................... 11 

LITERATURE CITED .................................................... 12 

APPENDIX 1 .......................................................... 17 

i 
LIST OF FIGURES 

Figure Page 

1 Line drawing of dolphin tooth in thin section 

showing distribution of tissues and structural 

anatomy ................................................. 13 

2 Decalcified and stained thin section of tooth 

from a female northern offshore spotted dolphin 

showing dentine and cementum............................ 14 

3 Decalcified and stained thin section of tooth 

from a six-year old female northern offshore 

spotted dolphin showing GLG components .................. 15 

4 Swatch from tooth thin section of a captive Hawaiian 

spinner dolphin imposed on left "shoulder"(HS) of 

tooth thin section of a northern offshore spotted 

dolphin, showing almost identical dentinal layering 

patterns ................................................ 16 

ii 
ESTIMATING AGE OF SPOTTED AND SPINNER DOLPHINS 

(STENELLA ATTENUATA AND STENELLA LONGIROSTRIS) FROM TEETH 

Albert C. Myrick Jr., Aleta A. Hohn, 

Priscilla A. Sloan, Makoto Kimura and Drew D. Stanley 

Southwest Fisheries Center 

National Marine Fisheries Service, NOAA 

La Jolla, California 92038 

ABSTRACT 

This paper is an account of preparation and examination techniques and 

criteria used to estimate age in decalcified and stained tooth thin sections 

from spinner and spotted dolphins. A dentinal growth layer group (GLG), 

composed of two thin light and two thicker dark-stained layers, is deposited 

annually. The GLG component layers are variably visible, but the "ideal" 

pattern and successive thinning of dentinal GLGs are used as a guide to 

determi ne GLG 1 imi ts. Age-speci f ic thicknesses of dentinal GLGs found in 

Hawaiian spinner dolphin teeth seem to be applicable to teeth of spotted 

dolphins and can be used as an aid in locating GLG boundaries. Cementa1 GLGs 

are composed of a dark-stained and a lightly stained layer and usually are 

deposited at a rate of one per year, but may be deposited every other year or 

two or three times per year. Two slightly different methods of counting 

dentinal GLGs are presented, along with guide1 ines for determi ning whether 

dentinal or cementa1 GLG counts provide the best estimate of age for a 

specimen. 

INTRODUCTION 

Age determination is a branch of research upon which dolphin population 

bi ol ogy depends heavi ly. Without a clear and accurate understandi ng of age 

composition, age at sexual maturity, age at first reproduction and natural 

longevity, the dynamics of a population cannot be determined. 

There are at least four requirements to determining age from growth layer 

groups (GLGs, terminology of Perrin and Myrick, 1980) in a dolphin tooth. 

First, one must be familiar with the timing of deposition and the distribution 

of dental tissues. Without knowledge of precisely where prenatal tissue ends 

and postnatal tissue begins, for instance, there can be no assurance that GLG 

counts are begun at the correct position in the tooth. 

Second, a reliable and effective system of preparing teeth that gives 

clear resolution of GLGs is necessary. Over the years, many techniques have 

been developed (see Perrin and Myrick, 1980, Report of the Workshop). Some 

have proven unsatisfactory in resolving GLGs easily (e.g., Perrin et al. 
2 

19771, while others provide excellent resolution of GLGs but are too time 

consuming or prohibitively expensive for large samples of teeth (e.g., Hohn, 

1980a). 

Third, to count GLGs with consistency, one must define in detail the 

structural pattern of a GLG as it appears in prepared teeth for a given 

species. The definition of layers counted for determination of age is vague 

in much of the literature. 

Finally, to relate GLG counts to age, the amount of time represented by a 

GLG should be demonstrated. With few exceptions (in which the results have 

been i nconcl usive, see references in Myri ck et a1 . MSl), age-determi nation 

studies have been unable to establish the timing of GLG deposition, largely 

because of a lack of suitable material. Commonly, GLGs as counted in most 

studies have been interpreted as yearly increments despite the lack of 

experimental support. 

Over the past several years the Marine Mammal Age Determination Project 

(SWFC, La Jolla, California) has conducted studies to improve techniques in 

preparation of thin sections of teeth for microscopic examination and to 

develop a real-time basis for estimating ages from dolphin teeth. This paper 

provides an account of preparation and exami nation techniques and the criteria 

developed to interpret age from the teeth of spotted and spinner dolphins, 

Stenel 1 a attenuata and Stenel 1 a longi rostri s. A study of seven captive 

Hawaiian spinner dolphins, S. longirostris, in which dental layers were 

calibrated using mu1 tiple taracycl i ne labels and periodic extractions of 

teeth (Myrick et al. MS1), forms the basis for definition of annual GLGs and 

age interpretation descri bed bel ow. No direct cal i brati on of dental GLGs has 

yet been possible for spotted dolphins or for spinner dolphins from the wild, 

but the GLG pattern in such samples closely resembles that of the captive 

sample. We have assumed that the time represented in GLGs in the captive 

spinner dolphins is the same in those of the wild samples of both species. 

DISTRIBUTION, DEPOSITION, AND UTILITY 

OF POSTNATAL DENTAL TISSUES 

Dental tissues in all delphinids have a common basic pattern of 

distribution and deposition (Fig. 1). The tooth of a new-born dolphin 

consists of a thin, external, conical mantle of enamel, formed prenatally, 

covering a thicker, basally-tapered cone of prenatal dentine and part of a 

thin, inner, layered cone of neonatal dentine. These internally-nested cones 

surround a central pulp cavity. 

lMyrick, A. C. Jr., E. W. Shallenberger, I. Kang, and D. B. MacKay. MS 

Cal i brati on of dental 1 ayers i n seven captive Hawai i an spi nner dol phi ns, 

Stenel 1 a longi rostri s, based on tetracycl i ne 1 abel s. 
3 

Denti ne 

The neonatal cone, i.e. the "neonatal line" in thin sections, is the 

first in a series of internally-deposited, layered cones comprising the GLG of 

postnatal dentine, the number of which is dependent upon the length of the 

dolphin's life and deposition rate. 

As a dolphin matures and dentine continues to accumulate at the wall of 

the pul p cavity , the pul p-cavi ty vol ume decreases gradual ly and successive 

GLGs become thinner. Typically, in regions of the late-formed dentine of 

ol der dol phi ns, the ti ssue shows i ncreasi ngly i r r e g u l a r GLGs. Farther inward, 

the dentine may be convoluted and layering may be indiscernible. The pulp 

cavity tends to be exceedingly narrow or occluded. If a dolphin lives beyond 

the time that these conditions commence, the dentine may not be useful for 

estimati ng the dol phi n ' s age accurately. 

Cemen turn 

Cementum is deposited postnatally; it is not known to occur in teeth of 

unborn dolphins. It is applied in concentric layers to the external surface 

of the root, below the "neck" of the tooth. Because its deposition is 

external and apparently unconfined, it is thought to form a continuous record 

representing a dolphin's entire life. Cementum, then, may be used to estimate 

maximum ages of older dolphins for which maximum dentinal age estimates are 

not possible. 

PREPARATION OF THIN 

SECTIONS OF TEETH 

Selection of Teeth 

In dolphins, the largest, most uniformly layered teeth occur in the 

central regions of the maxillary and mandibular tooth rows (Hui, 1978). 

Dentinal layering may cease earlier in maxillary teeth than in teeth of the 

mandible (Gurevich et al. 1980); therefore, teeth from the center of the lower 

tooth row are recommended for use in age determination (Perrin and Myrick 

1980, p. 40). The standard sampling procedure we use for spotted and spinner 

dolphins is to remove a four or five-cm-long section containing approximately 

eight to ten teeth from the middle of the dental series in the left mandibular 

ramus. Such sampling provides an adequate supply of teeth in case repeated 

preparations for a specimen become necessary. Any tooth from the mandibular 

section may be chosen for preparation. It is removed by breaking the alveolar 

bone away from the tooth. Utmost care is exercised to insure that the tooth 

is not chipped or otherwise damaged during removal. 

C1 eani ng 

Soft tissue is scraped from the tooth to be prepared with a scalpel or 
4 

fingernail after the tissue has been made pliable by soaking in water. Care 

is taken to insure that the cementum is not damaged during soft-tissue 

removal. 

Label s 

Each tooth is accompanied by an identification label of pure, cotton 

fiber bond through every step of the preparation procedure. The specimen number is recorded in soft lead pencil. 

Containers 

Each tooth, with its label, is placed in a clear plastic, hinged 

container 2.8 cm on each side. To allow the free flow of fluids through the 

container, all sides are perforated with a teasing needle, or other fine metal 

probe, that has been heated. The holes must be small enough to prevent thin 

sections of teeth from being lost. 

Decal c i f i cat i on 

The teeth are decalcified in RD02, a commercial rapid decalcifying agent 

containing hydrochloric acid as the principal active ingredient. 

Approximately 10 spotted dolphin teeth, each in its own container, may be 

decalcified at one time in 500 ml of RDO. (Species in which teeth are larger, 

e.g., Tursiops truncatus, require a proportionately larger volume of RDO per 

tooth .I To increase ci rcul ation , contai ners are agitated in the sol ution 

every 1-3 hours. Decalcification times vary with the age of the animal , because the thickness in the tooth is a function of age, and with the speciesspecific size of tooth. For spotted dolphins, teeth of the youngest animals, 

such a neonates and calves, require only 1-3 hours decalcification. These 

from body-1 ength classes 140-150, 150-160, 160-170, and 170-180 cm can require 

3-5, 6-8, 9-13, and 11-13 hours respectively. Teeth of the oldest animals, 

180-190+ cm in body length, require 11-15 hours for full decalcification. The 

500-ml volume of decalcifier tends to lose strength after 20 hours of 

continuous use on 10 specimens at a time. 

When decalcification is complete, the tooth is flexible and slightly 

translucent. A fully decalcified tooth may be pierced to the center without 

resistance with a fine, sharp needle, but this test disrupts the tissue of 

small teeth and is not recommended for routine use. Undue flexing of the 

tooth may damage internal tissues. Overdecalcification in RDO a1 so causes 

damage and renders the tooth useless for age determination. 

2Reference to trade names does not imply endorsement by the National Marine 

Fisheries Service, NOAA. 
5 

Complete removal of acid from the teeth is necessary to prevent RDO from 

interferinq with staining. After decalcification, the teeth are rinsed in 

running tap water by placing the containers in a large beaker under a 

faucet. The water flow causes the containers to tumble and optimizes 

circulation through the containers. Rinsing time is at least 3 hours. 

Sectioning 

Thin sections are cut with a sledge-type freezing microtome. A 

decalcified tooth is mounted on the cutting stage of the microtome, with the 

knife-blade set at about a 65" angle to the long axis of the tooth, and frozen 

with liquified C02. Serial longitudinal thin sections, 24 pin thick, are cut 

and discarded until the pulp cavity is revealed. Sectioning is continued 

until the entire extent of the pulp cavity is encompassed and thin sections 

cut subsequently fail to show the central cavity. This process yields a 

maximum number of near-center thin sections, which are returned to the 

container and rinsed in tap water to remove any residual RDO. The rest of the 

tooth is removed from the cutting stage and discarded. 

Staining 

The container of thin sections is immersed in Mayer's hematoxylin (see 

Appendix I for formula) for up to 30 minutes, until adequately stained. 

Excess stain is removed by a tap-water rinse. To enhance the contrast of the 

stained layers, the thin sections are "blued" in a 0.5% solution of ammonia 

water for 2-3 minutes. Prolonged exposure to the ammonia will reverse the 

stain. After blueing, the sections undergo a tap-water rinse for several 

minutes. 

Glycerin exchange 

Stained thin sections retain their color best when mounted in pure 

glycerin. Thin sections and label are first transferred from the container to 

a petri dish containing 1:l solution of glycerin and distilled water to soak 

for 5-10 minutes. Then they are placed in a petri dish of 100% glycerin to 

complete the exchange of glycerin for water in the thin sections. 

Selection of thin sections 

The petri dish is placed on the stage of a dissecting microscope and the 

thin sections to be mounted are selected. Ideally, those selected should be 

mid-1 ongi tudi nal . Each section should have a sharply-poi nted apex. The 

dentinal matrix should be light blue, with contrasting darkly-stained and 

1 i ghter stained 1 ayers appearing as sharply-poi nted chevrons a1 ong the midline. Changing the focal depth by adjustments of the focusing knob of the 

microscope shoul d produce only sl i ght re1 ati ve di spl acement of 1 ayers and pul p 

cavity margi ns. The cementum shoul d reveal more deeply-stai ned thin 1 ayers 

against a light blue background. Thin sections without a pulp cavity, those 
6 

stained too dark or too light, or cut off mid-center are not used. 

Mounting 

Several thin sections are placed in 100% glycerin on a slide. Wrinkles 

are smoothed out with a small blunt instrument so that each specimen lies 

flat. Excess glycerin is drained from beneath the cover slip, and cover-slip 

margins are cleaned with ethanol and sealed to the slide with Permount*. 

Specimen numbers and coded preparation information are recorded in waterproof 

ink on the frosted glass surface of the slide. The finished slides are placed 

on racks to dry. 

Appearance of the mounted thin section 

The thin mantle of enamel originally covering the apex of the tooth is 

absent, having been dissolved completely during decalcification. The prenatal 

denti ne occurs as a darkly-stained, re1 ati vely unl ayered chevron, thickest at 

the apex, with two tapered legs pointed basally. Adjacent and internal to the 

prenatal dentine, the neonatal line appears as a thin, distinctly unstained 

chevron. The remainder of the postnatal dentine occurs as a series of 

i nternal ly-nested, el ongate chevrons of decreasing thickness with a1 ternati ng 

darkly-stained and 1 ightly-stained layers (Fig. 2A). 

Cementum occurs along both sides and at the base of the root external to 

the denti ne. Its pattern consists of a1 ternati ng darkly-stai ned and 1 i ghtlystained 1 ayers oriented subparall el to the root and of f-1 apped basal ly 

(Fig. 26). 

DEFINING DENTINAL GLGs 

GLG appearance 

Ideally, each of our GLGs, (which in these species we have calibrated as 

representing one year-- see above), consists of an external, thin, lightlystained or unstained boundary 1 ayer (begi nni ng with the neonatal 1 i ne), 

followed in succession pulpwards by a thick, dark-stained layer, another thin, 

1 i ghtly-stai ned mi d-GLG 1 ayer (usual ly less di sti nct than the boundary 1 ayer) , and a second thick, darkly-stained layer. A GLG is considered to be complete 

when it contains all four layers (Fig. 3). 

Commonly, however, the four components are variably visible. In most 

regions of the first several GLGs, the two thick darkly-stained components may 

be light near the mid-GLG layers and darkest at the margins near the boundary 

layers. This gives the GLG an appearance of containing only the light 

boundary 1 ayer and one broad 1 ayer that is stained sl i ghtly darker. 

Exami nation of the GLGs through thei r enti re 1 ongi tudi nal extent, especi a1 ly 

in the apical region where components may be followed basally, often permits 

identification of all or some of the layers. Usually, at least the boundary 
7 

layer or the contrasting dark margin of an adjacent darker-stained layer is 

recognizable. Such landmarks are most reliable in delimiting a GLG. Despite 

the problem that all GLG components are not equally apparent, we have found 

that the "ideal" pattern and the progressive thinning of the GLGs pulpwards 

are important aids in defining GLGs. For adjacent GLGs, the later-formed GLG 

should be the same thickness or thinner than the earlier-formed GLG. 

The eighth to sixteenth GLGs are almost uniform in thickness, less 

vari ab1 e in component pattern, and easier to di sti ngui sh. Usual ly , thei r 

boundary layers or strong, dark adjacent margins are sharply demarcated. 

Age-speci f ic thickness of GLGs 

Studies of tetracycl i ne-1 abel ed teeth of seven Hawai i an spi nner dolphins, 

- S. longirostris, have shown that each GLG (as defined above) has a rather 

specific thickness and that the thickness of a given GLG varies little between 

teeth of the same individual or between teeth of different individuals (Myrick 

et al. MS1 1. GLG-thickness measurements were made perpendicular to the long 

axis of the teeth, from the base of the neonatal line, inwardly and downwardly 

at a 20 to 40" angle to the wall of the pulp cavity. Measurements made on the 

1 abel ed Hawaii an spinner teeth yielded the foll owing thicknesses for GLGs: 

First GLG 

Second I' 

Thi rd 'I 

Fourth 'I 

Fi f th 'I 

Sixth 'I 

Seventh I' 

Eighth 'I 

*Sample sizes o 

z S 

240.0 fm 0 

238.6 3.8 

171.0 'I 11.0 

145.0 'I 10.5 

119.6 'I 8.4 

95.4 'I 16.5 

87.0 'I 5.6 

71.3 I' 10.3 

N* 

7 

7 

7 

6 

6 

6 

6 

4 

z 

Ninth GLG 61.3 pm 

Tenth 'I 58 . 1 'I 

Eleventh " 55.0 I' 

Twelfth 'I 65.0 I' 

Thirteenth 'I 55.0 'I 

Fourteenth 'I 40.0 I' 

Fifteenth 'I 40.0 " 

Hawai i an spi nner dol phi ns used. 

S N* 

2.5 4 

2.4 4 

5.0 3 

1 

- 1 

1 

- 1 

- 

Aae-SDecific measurements of GLG thickness have been shown to have some 4, 

constancy in S. longirostris (Perrin et al. 19771, Pontoporia blainvillei; 

(Kasuya and Brownell , 1979) , and Tursiops truncatus (Hohn, 198 Ob 1 , a1 though 

neither labeled nor known-age specimens wer *e studied. 
8 

Constancy of GLG thickness for spotted and spinner dolphins 

GLGs in wild northern offshore spotted dolphins appear to have the same 

component-1 ayer pattern and thicknesses as those i n the Hawaii an spi nner 

dolphins studied by Myrick et al. (MS1). As shown in Figure 4, when the 

dentine from one of the captive Hawaiian spinner dolphins is compared with 

that of a wild spotter dolphin the resemblance between the two patterns is 

striking. When the GLG measurements determi ned from the capti ve sample were 

applied to the dentinal pattern of spotted dolphins, they corresponded 

approximately with the boundary layers of each of the first 15 GLGs. By 

continuing to apply the same 1 ast measurement on subsequent GLGs, boundary 

layers for up to the eighteenth GLG could be identified in cases where 

1 ayering remained undi storted. Despite the problem that experimental work on 

time calibration of dentinal layering has been possible only for the captive 

Hawai i an spi nner dol phi ns , unti 1 known-age or 1 abel ed spotted dol phi ns become 

available, we assume that a GLG represents the same amount of time in spinner 

and spotted dol phi ns. 

Extrapolating in this way provides a method for definition of GLGs in 

spotted dol phi ns. Nevertheless , because some vari ati on occurred in GLG 

thickness between i ndi vi dual spinner dol phins studied and because the degree 

of individual variation is unknown for spotted dolphins, the series of 

measurements is recommended only as an aid in locating GLG boundaries. 

Methods used to count dentinal GLGs in spotted dolphins 

To identify and count dentinal GLGs in the teeth of spotted dolphins, two 

slightly different methods have been used (by Hohn(AAH) and Myrick(ACM)). 

a. Method l(AAH). The whole tooth section is first inspected to 

determine the general pattern of layer groups. Then the layer groups are 

examined to define GLGs. Use is made of the "ideal" GLG pattern of component 

layers, depending heavily on the position and spacing of the dark margins of 

the darker-stained layers and on the progressive narrowing of layer groups 

pulpwards. The first two GLGs are identified using the prenatal dentine as a 

guide. Each of these GLGs is approximately the thickness of the prenatal 

dentine measured at a point about two-thirds of the distance down from the 

apex to the basal end of the prenatal wedge. All GLGs are examined throughout 

their longitudinal extent before making final interpretations. 

The chief advantages of this method are that: 1) it does not require an 

external aid that may affect interpretations if applied rigorously, 2) no 

single region is used principally to locate boundaries, and 3) GLG distortion 

has a minimal effect on final interpretations. However, locating boundaryand other component-layers may be difficult in regions with faint dentinal 

layers and some GLGs may be overlooked, resulting in an understimate of age. 

The relative order of magnitude of decreasing GLG thicknesses is evaluated 

without the aid of quantified reference points. In addition, the method is 

not extrapol ated from real -age studies of re1 ated species . 
9 

b. Method 2(ACM). The whole tooth section is inspected to characterize 

the pattern of layer groups. Although not applied rigorously, the series of 

GLG measurements derived from the study of Hawaiian spinner dolphins is used 

as an aid in locating GLG boundaries. The segment of dentine enclosed by each 

successi ve measurement is i nspected for GLG-component 1 ayers throughout its 

longitudinal extent and in relation to the general layering pattern of the 

whole tooth. Adjustments to the "standard" thicknesses are made on the basis 

of the general dentinal pattern and specific layer-component patterns observed 

relative to the "ideal" pattern. The first measurement inward of 240 pm from 

the prenatal edge of the neonatal line, taken at a point near the base of the 

prenatal dentinal wedge, gives the approximate distance of the external, thin, 

lightly-stained boundary layer of the second GLG. A 240-pm measurement from 

the adjusted boundary layer gives the approximate distance to the external 

boundary of the third GLG. Successive GLG boundaries are located using the 

appropriate standard thicknesses as a guide. 

The advantages of the method are that: 1) progressive thinning of GLGs 

can be quantified, aiding decisions about the limits of successive GLGs, 2) it 

provides a means of locating GLGs in faintly stained regions with relative 

ease, 3) it is extrapolated from GLG thicknesses in a related species with a 

simi 1 ar dentinal pattern for which real time was determi ned. Nevertheless, in 

applying the method rigorously to define GLGs, identification of component 

layers may tend to be forced by anticipation of the pattern. This is 

especially true in cases where layers are variably visible or compressed and 

indistinct. In addition, GLG thicknesses are not uniform from apex to base. 

Natural regional distortion in a tooth or imposed distortion from off-center 

sections affect GLG thickness, causing variability to be high. 

Differences between the methods 

Method 1 uses dark-stained boundary layers. Method 2 uses light boundary 

layers. Method 1 uses no external aid, but does use the prenatal thickness to 

estimate the limits of each of the first two GLGs. Method 2 uses the 

measurements as a guide. Method 1 was developed without extrapolation from 

real-time documentation of GLGs. Method 2 is aided by real-time documentation 

of GLG thickness in teeth of similar pattern representing a related species. 

Simi 1 ari ties between the methods 

Despite the differences , both methods are basical ly similar. GLGs are 

defined with reference to the same "ideal" pattern and progressive GLG 

thinning. Both methods require inspection of the whole tooth to identify 

general layer group patterns. Both rely on position and spacing of boundary 

layers. Both methods have resulted in the determination that the first two 

GLGs are of simi 1 ar thicknesses. Finally, both evaluate subsequent 1 ayer 

groups subjectively after the regular layeri ng pattern ceases. 
10 

CEMENTUM 

In general, each cemental GLG consists of two components: a darkly 

stained layer and a lightly stained layer. The problem with such a simple 

definition is that although the number of cemental GLGs often may equal the 

number of dentinal GLGs counted, they are not always equal. There is no 

regular thickness for a cemental GLG as there a dentinal GLG. In 

several regions along the tooth, cementum may contain GLGs in multiples, 1.e. 

with two or three times the number of GLGs observed in the dentine. In other 

regions it may be thin, with no distinct layering or with only half the number 

of GLGs observed in the dentine. Myrick et a1 . (MS)1 have made similar 

observations in Hawai i an spi nner dol phi n teeth usi ng tetracycl i ne 1 abel s to 

calibrate cementum and dentine. 

SUGGESTED PROTOCOL FOR DETERMINING 

AGE FROM GLGs 

The finding that cemental GLGs may occur in multiples or halves and the 

problems with pul p-cavi ty occlusion or layer indi sti nctness in denti ne point 

up the hazards of using either tissue independently for age determination. 

Before analyzing the layering pattern of cementum, we recommend that one 

should first evaluate the dentine and condition of the pulp cavity. In teeth 

with clear, regularly-formed dentinal GLGs and with an open pulp cavity, 

dentinal GLG counts should be relied upon for estimating age. The cemental 

count may be used to check the count from dentine; it should be the same as, 

or half, or exact multiples of the dentinal count. In other cases, cementum 

should be relied upon for the best estimation of age, with the best dentinal 

count used to evaluate whether the cemental GLGs were deposited as multiples 

or halves in the cementum. 

Speci f ical ly , we recommend the fol lowi ng protocol : 

1. If the pulp cavity is open: 

a. and regularly layered dentine is present throughout the tooth, the 

dentinal count should b e used as the best indicator of age; the 

cemental count should be used to check the dentinal count. If the 

cemental count is half, two times, or three times the dentinal 

count, assume that the cementum has been deposited as halves or 

multiples of the dentinal GLGs. 

b. and irregularly layered dentine is present, preventing a complete 

count of dentinal GLGs. count the dentinal GLGs as fully as 

possible, then count the -cementa1 GLGs. Use the number of cementa1 

GLGs as the age if it is less than or more than an exact multiple 

of the dentinal count. If it is equal to a multiple of the 

dentinal count, use the dentinal count as the age. If the maximum 

cemental count is much less than, but greater than half of the 

dentinal count, assume that cemental GLGs are halves and double the 

number to estimate age. If the cementum is unlayered or 

indistinctly layered, the only age estimate available is a minimum 

age as indicated by the dentine. 
11 

2. If pu 

a. 

b. 

p cavity is narrow or almost occluded: 

and regularly layered dentine occurs throughout the section , use 

the same procedure as in la. An exception is in cases where the 

cemental count is more than the dentinal count but less than a 

multiple or more than half of the dentinal count, then use the same 

procedure as in lb. 

and irregular dentine is present, use the same procedure as in 

Ib. 

3. If pulp cavity is closed or is filled with irregularly layered dentine, 

use the same procedure as lb. 

Since all of the criteria for final determinations of age rest chiefly on 

the accuracy of the dentinal counts, it is essential that careful evaluation 

of the pulp-cavity condition and scrupulous definition of dentinal GLGs be 

carried out. A tooth in which the pulp cavity is interpreted incorrectly as 

being closed and in which the dentinal GLGs are miscounted, may lead one to 

use the count of cemental GLG multiples as the estimated age. 

ACKNOWLEDGEMENTS 

We thank T. Kasuya, H. Marsh, D. E. Sergeant, D. DeMaster, D. Chapman, A. 

Wild, D. Siniff, F. Hester, R. L. Brownell, Jr., and W. F. Perrin for their 

critical reviews of the manuscript. H. Orr assisted in preparation of the 

figures. H. Becker typed the manuscript. 
12 

LITERATURE CITED 

Gurevich, V. S., B. S. Stewart, and L. H. Cornell. 1980 C198ll. The use of 

tetracycl i ne in age determi nation of common dol phi ns, Del phi nus 

del his In: W. F. Perrin and A. C. Myrick, Jr. (eds.) Age determination 

hhehwhales and sirenians. Rep. int. Whal. Commn (Special Issue 

"0. 31, 229 PHohn, A. A. 1980a C198ll. Analysis of growth layers in the teeth of Tursiops 

Perrin and A. C. Myrick, Jr. (eds.) Age determination of tootEd whales 

truncatus using light microscopy, microradiography, and SEM. In: W .. t 

and sirenians. Rep. int. Whal. Commn (Special Issue No. 31, 229 PHohn, A. A. 1980b. Age determination and age related factors in the teeth of 

western north Atlantic bottlenose dolphins. -- Sci. Rep. Whales -- Res. Inst. 

32 : 39-66. 

Hui, C. A. 1978. Reliability of using dentin layers for age determination in 

U. S. Dept. Comm. Nat. Tech. Inf. Ser. Publ. No. 

Kasuya, T. and R. L. Brownell, Jr. 1979. Age determination, reproduction, 

and growth of the Franciscana dolphin, Pontoporia blainvillei. - Sci. &. 

Whales Res. Inst. 31:45-67. 

Perrin, W. F., D. B. Holts, and R. B. Miller. 1977. Growth and reproduction 

of the eastern spinner dolphin, a geographical form of Stenella 

longirostris in the eastern tropical Pacific. Fish. Bull .7UXT 

Ts0.725. 

-- 

*- 

-- 

Perrin, W. F. and A. C. Myrick, Jr. (eds.) 1980 C198ll. Age determination of 

toothed whales and sirenians. Rep. int. Whal. Commn (Special Issue No. 

31, 229 p. 
13 

CF 

N 

R 

Enamel (substanti 

- 

Neonolal Line 

Prenatal Dentine 

Postnarol Dentine 

- 

Cementum (subsiantia 

IO adan mn itina) 

Orthodentine 

’ (substantia eburneo) 

C issea) 

Fig. 1. Line drawing of dolphin tooth in thin section showing distribution 

of tissLies and structural anatomy (from Perrin and Myrick, 1980). 
14 

A 

6 

Fig. 2. Decalcified and stained thin section of tooth from a female northern 

offshore spotted dolphin (specimen no. SWM 0001) viewed in plain 

transmitted 1 i ght (composi te photograph). 

A. Dentine showing 11 GLGs. (39x1 N=neonatal line. 

B. Cementum showing 11 GLGs. (150x1 
15 

uw 

200 pm 

Fig. 3. Decalcified and stained thin section of tooth from a six-year-old 

female northern off shore spotted dol phi n (specimen no. SER 0059) 

showi ng six denti nal GLGs (composi te photograph). 

two dark-stained layers (arrows) (plain transmitted light, 39x1. 

Each GLG contai ns 
16 

Fig. 4. Swatch from thin section of tooth of a captive Hawaiian spinner 

dolphin (specimen no. SWFC 104) imposed upon left "shoulder"(HS) of 

thin section from a northern offshore spotted dolphin (specimen no. 

WHB 0025) taken from the wild, showing almost identical dentinal 

1 ayeri ng patterns (composite photograph 1. 
17 

APPENDIX 1. Formula for Mayer' s hematoxyl in 

(Modified by Helen Gruel 

Hematoxylin 

Distilled water 

Sodi um iodate 

Aluminum potassium sulfate (alum) 

1.0 gm 

1000.0 cc 

0.1 gm 

50.0 gm 

Dissolve hematoxylin in water, using gentle heat if necessary. Add the sodium 

iodate, then the alum. Stir until the alum is dissolved. Store stain in the 

refrigerator to retard overripening. Stain may be used immediately after 

preparation. 
RECENT TECHNICAL MEMORANDUMS 

Copies of thisand other NOAATechnical Memorandums are available from the National Technical 

Information Service, 5285 Port Royal Road, Springfield, VA 221 67. Paper copies vary in price. Microfishe copies cost $3.50. Recent issues of NOAATechnical Memorandumsfrom the NMFS Southwest 

Fisheries Center are listed below: 

NOAA TM-NMFS SWFC 20 

21 

22 

23 

24 

25 

26 

27 

28 

29 

Testing methods of estimating range and bearing to cetaceans 

T. D. SMITH 

(1 982) 

"An annotated bibliography of the ecology of co-occurring tunas 

(Katsuwonus pelamis, Thunnus albacares) and dolphins (Stenella 

attenuata, Stenella longirostris and Delphinus delphis in the 

eastern tropical Pacific" 

aboard the R/V D.S. Jordan. 

S. D. HAWES 

(November 1982) 

Structured flotsam as fish aggregating devices. 

R. S. SHOMURA and W. M. MATSUMOTO 

(November 1982) 

Abundance estimation of dolphin stocks involved in the eastern 

tropical Pacific yellowfin tuna fishery determined from aerial and 

ship surveys to 1979. 

R. S. HOLT and J. E. POWERS 

(November 1982) 

Revised update and retrieval system for the CalCOFl oceanographic data file. 

L. EBER and N. WILEY 

(December 1982) 

A preliminary study of dolphin release procedures using model 

D. B. HOLTS and J. M. COE 

(December 1982) 

"Possible effects of sampling biases on reproduction rate estimates 

T. POLACHECK 

(January 1983) 

"Reports of porpoise experiment testing detection of on-track 

R. S. HOLT 

(February 1983) 

"Two computer programs to project populations with time-varying 

T. GERRODETTE, D. GOODMAN 81 J. BARLOW 

(February 1983) 

Report of eastern tropical Pacific research vessel marine mammal 

R. S. HOLT 

(March 1983) 

purse seines. 

for porpoise in the eastern tropical Pacific." 

schools (pet dots), March 7-April 5, 1981 ." 

vital rates." 

survey, May 15-August 3, 1982.