Journal of Threatened Taxa | | 26 September 2022 | 14(9): 21805–21810


ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print)

#7727 | Received 26 October 2021 | Final received 10 March 2022 | Finally accepted 14 August 2022


Age estimation of Tiger Panthera tigris (Linnaeus, 1758) and Lion Panthera leo (Linnaeus, 1758) (Mammalia: Carnivora: Felidae): applicability of cementum annuli analysis method


Vipin 1  , Chandra Prakash Sharma 2 , Vinita Sharma 3 , Surendra Prakash Goyal 4 , Heather Stevens 5 & Sandeep Kumar Gupta 6


1,2,4,,6 Wildlife Institute of India, Dehradun, Post Box No.18, Chandrabani, Dehradun, Uttarakhand 248001, India.

1,5 DeerAge, Wildlife Analytical Laboratories, 2814, Brook Street No. 114, Missoula, Montana 59801, USA.

3 Department of Zoology, Central University of Jammu, Rahya-Suchani (Bagla), District, Samba, Jammu, Jammu & Kashmir 181143, India.

1, 2, 3, 4,
5, 6 (corresponding author)


Abstract: We describe the applicability of the cementum annuli analysis technique for estimating the age of Tiger Panthera tigris and Asiatic Lion Panthera leo using incisor teeth. We used I2 and I3 incisor teeth from the right mandible of Tiger and I2 and I3 from the left premaxilla of the Lion. The longitudinal sections of the teeth were prepared using an economical hand grinding technique with the help of sandpaper, followed by decalcification and staining with hematoxylin. Two cementum layers were observed under the microscope in each of the I2 and I3 incisor teeth of the Tiger and six cementum layers were observed in each of the I2 and I3 incisor teeth of the Lion. The permanent incisors in Tiger and Lion erupt between 12 and 14 months of age; hence, we added one year to the counted number of cementum layers to estimate the final age of Tiger and Lion incisors. The age of Tiger and Lion incisors were estimated to be of three years and seven years, respectively. This method may be suitable for estimating other carnivores’ age and applicable in wildlife forensic studies.


Keywords: Big cats, carnivore, epoxy, grinding, incisor, premolar, teeth, wildlife forensics.


Editor: Angie Appel, Wild Cat Network, Bad Marienberg, Germany.     Date of publication: 26 September 2022 (online & print)


Citation: Vipin, C.P. Sharma, V. Sharma, S.P. Goyal, H. Stevens & S.K. Gupta (2022).  Age estimation of Tiger Panthera tigris (Linnaeus, 1758) and Lion Panthera leo (Linnaeus, 1758) (Mammalia: Carnivora: Felidae): applicability of cementum annuli analysis method. Journal of Threatened Taxa 14(9): 21805–21810.


Copyright: © Vipin et al. 2022. Creative Commons Attribution 4.0 International License.  JoTT allows unrestricted use, reproduction, and distribution of this article in any medium by providing adequate credit to the author(s) and the source of publication.


Funding: The study was funded by the Wildlife Institute of India, Dehradun


Competing interests: The authors declare no competing interests.


Author details: Vipin is working as a Director of Research at DeerAge, Missoula, Montana, USA. His areas of research interest are wildlife conservation, age estimation, wildlife forensics and disease diagnosis. Chandra Prakash Sharma is working as a Senior Technical Officer, Wildlife Forensic and Conservation Genetics Cell, Wildlife Institute of India, Dehradun. He is dealing with wildlife offence cases with morphological and other relevant techniques. Along with the primary task of developing new protocols for the identification of species from wildlife parts, he is involved in teaching and training wildlife enforcement officials in curbing illegal wildlife trade and morphological identification of wildlife articles.  Vinita Sharma is an Assistant Professor in the Department of Zoology, Central University of Jammu, Jammu and Kashmir. Her area of research is related to animal taxonomy, systematics and behavior, wildlife and conservation biology, human-wildlife conflict, wildlife forensics, comparative anatomy and geometric morphometrics. Surendra Prakash Goyal was Scientist G and now working as a subject matter specialist at the Wildlife Institute of India, Dehradun. His research areas include wildlife ecology, field research methods, wildlife-habitat relationships of ungulates and carnivores, estimation of food habits, habitat fragmentation and corridors, use of lab methods, especially in nutritional ecology, molecular ecology, landscape genetics. Heather Stevens is heading Nationwide Histology Inc providing the highest quality service for research clients and the DeerAge, Wildlife Analytical Laboratories, dedicated to quality age estimation of game animals across the United States of America. Sandeep Kumar Gupta is Scientist E, Head, Department of Animal Ecology and Conservation Biology and Nodal Officer, Wildlife Forensic and Conservation Genetics Cell, Wildlife Institute of India, Dehradun. His areas of research interest are conservation genetics, wildlife forensics, evolutionary genetics of rare and endangered species. He is also undertaking teaching and training courses on illegal wildlife trade.


Author contributions: V, CPS, SPG & SKG conceived the study. V, CPS & VS carried out the laboratory work. V & HS did the data analysis. V, VS & SKG wrote the article. CPS, SKG, SPG & HS reviewed the article.


Acknowledgements: We gratefully acknowledge the support of the director and dean, Wildlife Institute of India, Dehradun.




The age of carnivores needs to be estimated in studies about the demography of species and for understanding population dynamics (Skalski et al. 2005; Foresman 2012), age class (Angerbjorn et al. 2004; Creel et al. 2004), population monitoring trends (Barthold et al. 2016), human-wildlife interactions (Conover 2002; Frank et al. 2005), and illegal wildlife trade (Williams et al. 2015). The widely used methods for age estimation of carnivores are assessments of tooth eruption (Slaughter et al. 1974), wearing of a tooth crown (Harris 1978; Stander 1997; Gipson et al. 2000), closure of pulp chamber (Marks & Erickson 1966; Zapata et al. 1997; Binder & Van Valkenburgh 2010), and cementum analysis (Klevezal & Kleinenberg 1967; Matson 1981; White & Belant 2016).

The method to estimate the age of the Tiger Panthera tigris has mostly been limited to assessing tooth eruption, wearing (Mazak 1979, 1981; Miles & Grigson 2003), and gum line recession (Fŕbregas & Garcés-Narro 2014). The methods described to assess the age of the Lion Panthera leo refer to sizes of body and mane, pigmentation on the nose, tooth wear (Schaller 1972; Smuts et al. 1978; Whitman et al. 2004; Whitman & Packer 2007; Ferreira & Funston 2010), closure of the pulp chamber (White & Belant 2016), the ratio of tooth areas (White et al. 2016), tooth eruption (Schneider 1959) and cementum analysis (Spinage 1976; Smuts et al. 1978; White & Belant 2016).

Amongst the various age determination methods available, the cementum analysis method has been recommended for its accuracy (Mundy & Fuller 1964; Marks & Erickson 1966; Klevezal & Kleinenberg 1967; Craighead et al. 1970; Willey 1974, Grue & Jensen 1979; Johnston et al. 1987; Matson et al. 1993; Mbizah et al. 2016; Vipin et al. 2018). The described technique does not need a costly microtome for tooth sectioning, so most of the items required are generally available in a standard lab (Vipin et al. 2018).

To date, studies on age estimation of South Asian mammals through cementum layer analysis are limited to Chital Axis axis (Vipin et al. 2018). Here we present the applicability of this method for estimating the age of Tiger and Asiatic Lion.



Material and Methods


We used Tiger mandible and Lion skull from Wildlife Forensic and Conservation Genetics Cell’s repository. We tested the applicability of the developed method to estimate the age of an incisor (I3) from the mandible of a Tiger seized in the illegal wildlife trade, which was sent to Wildlife Institute of India, Dehradun, for species confirmation.


Sample collection and preparation of longitudinal section of teeth

Canines of Tigers and Lions are in high demand compared to other species’ teeth in the illegal wildlife trade. Hence, in comparison to other types, the chances of their availability for determining age are limited. In both species, the permanent incisors number is six times more than premolar (PM2), which is a plus point if some tooth gets damaged during processing for cementum analysis. Therefore, we selected incisors in the current study and based on the availability of their types, the incisors were extracted.

Two permanent incisor teeth (I2, I3) out of three were used from the right mandible of a Tiger (Image 1A, B, C) and two incisors (I2, I3) from the left premaxilla of a Lion. The teeth from the Tiger mandible were extracted by boiling it in water for ten minutes, after which they detached easily from the mandible. From the Lion premaxilla, the teeth were removed with the help of pliers with utmost care so that the periodontal membrane remained intact. We used the protocol described by Vipin et al. (2018) for preparing the longitudinal sections of the incisor teeth with a thickness of around 57 µm with steps, as shown in Image 2. We then used a Leica DMR microscope to examine the tooth sections.


Calculation of age from cementum annuli

In felids, all permanent incisors except I3 erupt before other teeth (Miles & Grigson 2003). In Tiger, the permanent tooth eruption starts between 8.8–9.5 months and completes at the age of 12–14 months (Mazák 1979, 1981). In Lion, permanent I1, I1, I2, I2 fully erupts between 9–11 months and I3 and I3 start erupting by the end of this period (Smuts et al. 1978). I3 and I3 completely replace their deciduous counterparts at the age of 12 to 14 months, while P2 starts erupting between this period in Lions (Smuts et al. 1978). Though no published data related to age estimation of Tiger using cementum analysis is available; many researchers have used permanent incisors, canine, and second premolar teeth to develop age estimation methods in Lions utilizing this technique (Smuts et al. 1978; Cheater 2006; White & Belant 2016). The time taken by different tooth types for their permanent eruption has been reported unequal in other species of carnivores and ungulates (Zapata et al. 1995; Azorit et al. 2004). In P2 of African Lions, it is established that the first rest is formed in the second year of age, so we had to add one to the counted number of cementum lines to estimate the final age (White & Belant 2016). We counted the acellular cementum layer in the root portion of the teeth, which is formed annually and stains dark with hematoxylin (Matson et al. 1993). Therefore, we added a minimum of one year in both species’ final age estimation. The presence of one cementum layer in a permanent incisor tooth of Tiger and Lion indicates that the animal has lived one year at least. The age of sectioned teeth in years was calculated according to the formula

Age in year = Number of cementum layers + 1 year

The cementum layers in the incisor teeth were photographed wherever these were seen distinctly and clearly.





The teeth sections of the Tiger showed two cementum layers for I2 (Image 3 A, B, C) and I3 (Image 4 A, B); thus, the Tiger’s age was estimated to be three years. The Lion had six cementum layers in I2 (Image 5A, B) and I3 (Image 6); therefore, Lion’s age was estimated to be seven years

We found two dark cementum layers on the Tiger’s incisor seized in the illegal wildlife trade; hence its age was estimated as three years (Image 7). Therefore, the developed method may also be applied to estimate the age of tigers in the illegal wildlife trade.




White & Belant (2016) used paired PM2 teeth for estimating the age of free-ranging African Lions of unknown age through cementum line count and showed that cementum layer count in PM2 is unsuitable for ageing Lions. Their analysis revealed that in 19 out of 31 PM2 pairs, the cementum line count differed by 1–2 lines and even increased to seven lines for other pairs. According to Smut et al. (1978), cementum lines in canine teeth of Lions complied significantly with their known ages. So to compare the results of White & Balent (2016) about Asiatic Lions, a large sample size of the PM2 teeth is needed, or more incisors or a different tooth type needs to be analysed for cementum layer count. In ungulates, the accuracy of age estimation through cementum analysis decreases with the age of the specimens (Hamlin et al. 2000). More research is necessary to assess whether this is also true for carnivores.

We recommend validating the current procedure while estimating age based on cementum layer count. Matson et al. (1993) suggested two main tests for validating the cementum analysis for estimating age, namely the “blind” duplicate test when two or more teeth are available and using a tooth of known age but without having the prior knowledge of its age. Teeth of known age were not available for both species; however, all incisor teeth showed clear and distinct cementum layers. The periodontal membrane in all studied teeth confirms that all cementum layers were present in the longitudinal sections. Hence, the current method can show all cementum layers clearly and distinctly in incisor teeth.

The same protocol may be applied to develop age estimation protocols for other mammal species.



For images—click here for full PDF.




Angerbjorn, A., P. Hersteinsson & M. Tannerfeldt (2004). Arctic Foxes: Consequences of resource predictability in the Arctic Fox—two life history strategies, pp. 164–172. In: MacDonald, D.W. & C. Sillero-Zubiri (eds.). Biology and Conservation of Wild Canids. Oxford University Press, New York, 450 pp.

Azorit, C., J. Muńoz-Cobo, J. Hervás & M. Analla (2004). Ageing through growth marks in teeth of Spanish Red Deer (Cervus elaphus hispanicus). Wildlife Society Bulletin 32(3): 702–710.

Barthold, J.A., A.J. Loveridge, D.W. Macdonald, C. Packer & F. Colchero (2016). Bayesian estimates of male and female African Lion mortality for future use in population management. Journal of Applied Ecology 53(2): 295–304.

Binder, W.J. & B. Van Valkenburgh (2010). A comparison of tooth wear and breakage in Rancho La Brea sabertooth cats and dire wolves across time. Journal of Veterinary Palaeontology 30(1): 255–261.

Cheater, A. (2006). Use of the upper second premolar for age determination of the African Lion (Panthera leo) in sub-saharan Africa, for purposes of remote monitoring. Tshwane University of Technology, Tshwane, South Africa.

Conover, M. (2002). Resolving human-wildlife conflicts: the science of wildlife damage management. Lewis Publishers, Boca Raton, Florida, 418 pp.

Craighead, J.J., F.C. Craighead & H.E. McCutchen (1970). Age determination of Grizzly Bears from fourth premolar tooth sections. Journal of Wildlife Management 34: 353–363.

Creel, S., M.G.L. Mills & J.W. McNutt (2004). African Wild Dogs: Demography and population dynamics of African Wild Dogs in three critical populations, pp. 337–350. In: Macdonald, D.W. & C. Sillero-Zubiri (eds.). Biology and conservation of wild canids. Oxford University Press, New York, 450 pp.

Fŕbregas, M.C. & C. Garcés-Narro (2014). Validation of gum-line recession as a reliable technique to age Tigers. European Journal of Wildlife Research 60(6): 947–950.

Ferreira, S. & P.J. Funston (2010). Age assignment to individual African Lions. South African Journal of Wildlife Research 40(1): 1–9.

Foresman, K.R. (2012). Carnivores in hand, pp. 130–51. In: Boitani, L. & R.A. Powell (eds.). Carnivore ecology and conservation: A handbook of techniques. Techniques in ecology and conservation. Oxford University Press, New York, 506 pp.

Frank, L.G., R. Woodroffe & M.O. Ogada (2005). People and predators in Laikipia District, Kenya, pp. 286–304. In: Woodroffe, R., S. Thirgood & A. Rabinowitz (eds.). People and Wildlife: conflict or coexistence? Cambridge University Press, New York, 517 pp.

Gipson, P.S., W.B. Ballard & R.M. Nowak (2000). Accuracy and precision of estimating age of Gray Wolves by tooth wear. Journal of Wildlife Management 64: 752–758.

Grue, H. & B. Jensen (1979). Review of the formation of incremental lines in tooth cementum of terrestrial mammals. Danish Review of Game Biology 11: 1–48.

Hamlin, K.L., D.F. Pac, C.A. Sime, R.M. DeSimone & G.L. Dusek (2000). Evaluating the accuracy of ages obtained by two methods for Montana ungulates. Journal of Wildlife Management 64(2): 441–449.

Harris, S. (1978). Age determination in the Red Fox (Vulpes vulpes) and evaluation of technique efficiency as applied to a sample of suburban foxes. Journal of Zoology 184(1): 91–117.

Johnston, D.H., D.G. Joachim, P. Bachmann, K.V. Kardong, R.E.A. Kardong Stewart, L. Dix, M.A. Strickland & l.D. Watt (1987). Ageing furbearers using tooth structure and biomarkers, pp. 228–243. In: Novak, M., J.A. Baker, M.E. Obbard & B. Malloch (eds.). Wild furbearer management and conservation in North America. Ontario Trappers Association, Toronto, 1150 pp.

Klevezal, G.A. & S.E. Kleinenberg (1967). Age determination of mammals from annual layers in teeth and bones. Severtsov Institute of Animal Morphology, Academy of Science of the USSR, Moscow, 128 pp. (in Russian)

Marks, S.A. & P.W. Erickson (1966). Age determination in Black Bear. Journal of Wildlife Management 30: 389–410.

Matson, G., L. Van Daele, E. Goodwin, L. Aumiller, H. Raynolds & H. Hristienko (1993). A laboratory manual for cementum age determination of Alaskan Brown Bear first premolar teeth. Matson’s Laboratory, Milltown, Montana, 52 pp.

Matson, G.M. (1981). Workbook for cementum analysis. Matson’s Laboratory, Milltown, MT, 30 pp.

Mazak, V. (1979). Der Tiger Panthera tigris. Second edition, Neue Brehm Bücherei, A. Ziemsen Verlag, Wittenberg Lutherstadt (GDR), 228 pp.

Mazák, V. (1981). Panthera tigris. Mammalian Species 152: 1–8.

Mbizah, M.M., G. Steenkamp & R.J. Groom (2016). Evaluation of the applicability of different age determination methods for estimating age of the endangered African Wild Dog (Lycaon pictus). PLoS One 11(10): e0164676.

Miles, A.E.W. & C. Grigson (2003). Colyer’s Variations and diseases of the teeth of animals. United Kingdom: Cambridge University Press, 692 pp.

Mundy, K.R.D. & W.A. Fuller (1964). Age determination in Grizzly Bear. Journal of Wildlife Management 28: 863–866.

Schaller, G.B. (1972). The Serengeti Lion: a study of predator–prey relations. University of Chicago Press, 504 pp.

Schneider, K.M. (1959). Zum Zahndurchbruch des Löwen (Panthera leo) nebst Bemerkungen über das Zahnen einiger andere Grosskatzen und der Hauskatze (Felis catus). Der Zoologische Garten 22: 240–361.

Skalski, J., K. Ryding & J. Millspaugh (2005). Wildlife demography: Analysis of sex, age, and count data. Elsevier Academic Press, Burlington, MA, 656 pp.

Slaughter, B.H. R.H. Pine & N.E. Pine (1974). Eruption of cheek teeth in Insectivora and Carnivora. Journal of Mammalogy 55(1): 115–125.

Smuts, G.L., J.L. Anderson & J.C. Austin (1978). Age determination of the African Lion (Panthera leo). Journal of Zoology 185(1): 115–146.

Spinage, C.A. (1976). Incremental cementum lines in the teeth of tropical African mammals. Journal of Zoology 178(1): 117–131.

Stander, P.E. (1997). Field age determination of Leopards by tooth wear. African Journal of Ecology 35(2): 156–161.

Vipin, V. Sharma, S.K. Gupta, C.P. Sharma, K. Sankar & S.P. Goyal (2018). Development of a fast and low-cost age determination method in Spotted Deer, Axis axis. Folia Zoologica 67(3–4): 186–197.

White, P.A., D. Ikanda, L. Ferrante, P. Chardonnet, P. Mesochina & R. Cameriere (2016). Age estimation of African Lions Panthera leo by ratio of tooth areas. PLoS One 11(4): e0153648.

White, P.A. & J.L. Belant (2016). Individual variation in dental characteristics for estimating age of African Lions. Wildlife Biology 22(3): 71–77.

Whitman, K., A. Starfield, H. Quadling & C. Packer (2004). Sustainable trophy hunting of African Lions. Nature 428 (6979): 175–178.

Whitman, K.L. & C. Packer (2007). A hunter’s guide to ageing Lions in eastern and southern Africa. Safari Press, 46 pp.

Willey, C.H. (1974). Ageing Black Bears from first premolar tooth sections. Journal of Wildlife Management 38: 97–100.

Williams, V.L., D.J. Newton, A.J. Loveridge & D.W. Macdonald (2015). Bones of contention: An assessment of the South African trade in African Lion Panthera leo bones and other body parts. TRAFFIC, Cambridge & WildCRU, Oxford, 128 pp.

Zapata, S.C., A. Travaini & M. Delibes (1995). Comparacion entre varias tecnicas de estimacion de la edad en Zorros, Vulpes vulpes, de Dońana (sur de la peninsula iberica). Dońana Acta Vertebrata 22: 29–50.

Zapata, S.C., R.G. Perea, J.F. Beltrán, P. Ferreras & M. Delibes (1997). Age determination of Iberian Lynx (Lynx pardinus) using canine radiograph and cementum annuli enumeration. Zeitschrift für Säugetierkunde 62: 119–123.