A PRELIMINARY STUDY ON THE METHOD OF TIME
MARKING WITH LEAD SALT AND
TETRACYCLINE ON THE TEETH OF FUR SEALTEIJI YAGI*, MASAHARU NISHIWAKI AND
MASAYUKI NAKAJIMA**Few studies have been so far reported on the technique of the age determination offur seal. Scheffer (1950) described that the number of growth ridges appearing
on the root of the canine tooth indicated the age of fur seal. Matsumoto (1958)
developed the age-reading technique on the basis that the number of the transparent zones of the dentine corresponds to that of the growth rings on the surfaceof the teeth.
Kubota et al. (1961) stated that the transparent zone observed in the longitudinal ground section of the dentine terminates at the top of the growth ridge on
the dental root. After histological studies, they have also found that the interglobular spaces, the granular layer of Tomes, and the transparent zone are formedin regular turn every year in the dentine.
The method of counting the number of annual rings of the contour or of thetransparent bands of the dentine is rather easy and this is in practical use for theyoung animal. But the method is more difficult in the case of older animal, because the annual rings deposited become closer together with age. In addition,some thiner transparent zones of the dentine appear occasionally between two
annually formed transparent den tines in many specimens especially of female. Oneof the practical difficulties and confusions in determining the age comes mainly
from these zones in the case of the ground section of the dentine.
The present paper is a preliminary study to clarify the developmental processof the dentine by recording the time by the following two methods of intravitalstaining for hard tissues. The first is the modified method with lead acetate devisedby Okada and Mimura (1938) and the second is with tetracycline adopted by Milchet al. (1957). Nishiwaki et al. (1953) reported that the method using lead acetate(dose of 5 mg of lead acetate per kg. of body weight by intramuscular injection)
is applicable for the time-marking of the dolphin-tooth.
As to the method using tetracycline, many studies so far reported show that
this antibiotic tends to combine with calcium when deposited. After administration of tetracycline to an animal an induced fluorescence can be detected in thetissues where tetracycline is distributed.
MATERIAL AND METHODSIn the present experiment a female fur seal of 25 kg. in body weight caught on* Institute for Hard Tissues, Tokyo Medical and Dental University.

* * Enoshima Aquarium.

191

192

T. YAGI, M. NISHIWAKI AND M. NAKAJIMA

June 16, 1959 in the water off Ozuchi, Iwate Prefecture, Japan, was used for thispurpose.
The animal received the iajections on August 27, 1959, after rearing about
two-month period in the pond of the Enoshima Marineland (an aquarium). An
injection containing lead complex salt was prepared by mixing lead acetate withsodium citrate in the rate of 1 : 3 and was administered intramuscularly into thenates. The dose was 5 mg of lead acetate per kg. of body weight. Tetracyclinehydrochloride prepared commercially was administered by a single muscular injection in the dose of 4 mg per kg. of body weight, followed for seven days by thesame dose contained in each of the twice daily diet. Therefore, the animal received
the total amounts of 1500 mg of tetracycline administered orally or parenterally.
The animal survived for 162 days after the initial injection. The canine teeth
which were removed from the dead animal were fixed in a 10 % neutralized formalin solution. The teeth were decalcified in the 0.2 N hydrochloric acid saturated
with hydrogen sulfide, and then cut into frozen sections. They were next immersed in a 0.1 % solution of gold trichloride then in a 5 % sodium thiosulfate
solution to obtain a gilded lead line.
In order to observe the localization of tetracycline undecalcified ground sections of the teeth, approximately 50 f1. thick, were put to microscopic examination
for induced fluorescence under ultraviolet illumination. The microphotographs
were taken with the aid of an orange filter.
RESULT AND DISCUSSION

The line made by the deposition of lead was found in the sections of the teeth
prepared by the procedure given above. Fig. 1 shows the deposited line in the
middle of the developing dentine. The fluorescent structure characteristic to thedeposition of tetracycline is shown in Fig. 2 in which the structure is apparently
demonstrated as bright yellow line or band under the ultraviolet light.
As shown in these figures the lines, both of lead acetate and tetracycline, arelocated in the position of the growth layer of the dentine which is formed practicallyat the same time as the development of the dentine. This portion of the dentine
confined between the marked line and the predentine corresponds to the increaseduring a five-month period from August 27 to February 2. The longitudinalground section of the tooth shows that the age of the fur seal is identified as approximately four years by counting the contour growth ridges or transparent
zones of the dentine (PLATE II-A).
In the sections of the dentine some peculiar lines presumably caused by metabolic disturbances in the development of the dentine were observed. The firstline, the outermost of the dentine, was recognized as the neonatal line. The lastone, coincided in position with the marking line, seemed to be caused by the administration of tetracycline (PLATE III). It is already known that the administration of tetracycline depresses the calcification either in skeleton or in tooth, whilelead acetate in the appropriate doses shows no effect in development of the calcifiedTIMEMARKINGO NTHETEETHOFFURSEAL

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T. YAGI, M. NISHIWAKI AND M. NAKAJIMAof Mr. Izumi Morisaki in the course of studying fluorescence.
SUMMARY

Intravital stammg after the modified lead acetate method and tetracycline formarking the time on the dentine of the fur seal was carried out, in order to establish
a reliable method for the age determination of this animal. A female fur seal
received the drugs and survived for I 62-day period.
1. Lead acetate was given intramuscularly in the dose of 5 mg per kg. ofbody weight. The iajection containing lead complex salt by adding sodium citrate
to lead acetate, produced deposited line of lead in the area of the dentine in which
matrix calcification took place. The lead line was demonstrable histochemicallyin the decalcified sections.
2. Tetracycline was given in the dose of 60 mg per kg. of body weight orally
and parenterally. The administration of this was divided into seven successive
days and given practically at the same time as the lead injection. The line oftetracycline-induced fluorescence was evident in the ground section of the dentine
under the ultraviolet illumination.
3. Two lines produced by deposition of lead and tetracycline were localized
in the same position of the dentine. The width of dentine enclosed by the marked
band revealed the increase of the dentine during a five-month period from
September to January.
4. It was estimated that the fur seal was about four years old both fromthose results and from the annual rings on the root and the dentine.
5. The methods described here may be adopted as a technique for the age
determination especially in the older animal in which the pattern of the annualrings is not well defined.
REFERENCES
BEVELANDER, G., RoLLE, G. K. and CoHLAN, S. Q. (1961). The effect of the administration of tetracycline
on the development of teeth. ]our. Dent. Res., 40: 1020-1024.
HARRIS, W. H. (1960). A microscopic method of determining rates of bone growth. Nature, 188: 10381039.
KUBOTA, K., NAGASAKI, F., MATSUMOTO, K. and TsuBOI, M. (1961). Histological studies on the growth layers
in the maxillary canines of fur seals as an indicator of age. Part 1. The teeth at the age of one to nine
year. Bull. Tokyo Med. Dent. Univ., 8: 261-285.
MATSUMOTO, K. (1958). Unpublished, manuscript.
MILCH, R. A., RALL, D. P. and TOBIE,]. E. (1957). Bone localization of the tetracyclines. ]our. Natl. Cancer
Inst., 19: 87-91.
MILCH, R. A., ToBIE,j. E. and ROBINSON, R. A. (1961). A Microscopic study of tetracycline localization inskeletal Neoplasma. ]our. Histochem. Cytochem., 9: 261-270.
NAGASAKI, F. (1961). Population study on the fur-seal herd. Special Publication, Tokai Regional Fisheries
Research Laboratory, 7: 1-60.
NISHIWAKI, M. and YAGI, T. (1953). On the age and the growth of teeth in a dolphin, (Prodelphinus
caeruleo-albus). (I). Sci. Rep. Whales Res. Inst., 8: 133-145.

TIME MARKING ON THE TEETH OF FUR SEAL

195

NrsmWAKI, M· and YAGI, T. (1954). On the age-determination method of the toothed whale by the studyof the tooth. Proc. Jap. Acad., 30 399-404.
NrsmwAKr, M., HrnrvA, T. and 0HSUMI, S. (1958). Age study of sperm whale, based on reading of toothlaminations. Sci. Rep. Whales Res. Inst., 13: 135-154.
OKADA, M. and MrMURA, T. (1938). Zur Physiologie und Pharmakologie der Hartgewebe. I. Mitteilung:
Eine Vitalfarbungsmethode mit Bleisalzen und ihre Anwendung bei den Untersuchungen itber die rhythmische Streifenbildung der harten Zahngewebe. Jap. ]our. Med. Sci., fl7 Pharmacology, 11 : 166-170.
SCHEFFER, V. B. (1950). Growth layers on the teeth of pinnipedia as an indication of age. Science, 112:309-311.
ScHOUR, I. and KRONFELD, R. (1939). Neonatal dental hypoplasia. ]our. Amer. Dent. Assn., 26: 18-32.

EXPLANATION OF PLATESPLATE I
Ventral view of the skull (A), dorsal view of the mandible (B), the left mandible canine (C)and the left maxillary canine (D) of the fur seal, 25 kg. weight female, used in this observation.
PLATE II
Longitudinal ground section of left maxillary canine of the fur seal (A) compared with aspecimen of 4-year-old female (B).
N: Neonatal line. T-1, 2, 3: Transparent zone of the dentine.
PLATE IIILongitudinal ground section ofleft maxillary canine of the fur seal, around 158 days after theadministration of tetracycline, under ultraviolet light (A) and under tungsten light (B).
TC : Fluorescent line of deposited tetracycline.
A: Line of auto-fluorescence.

T. YAGI,M. NISHIWAKIAND M.NAKAJIMA

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