Age determination and establishment of reproductive condition from marine mammal biopsy sampling

Published byDepartment of ConservationPO Box 10-420Wellington, New Zealand DOC Science Internal Series is a published record of scientific research carried out, or advice given,by Department of Conservation staff or external contractors funded by DOC. It comprises reports andshort communications that are peer-reviewed.
Individual contributions to the series are first released on the departmental website in pdf form.
Hardcopy is printed, bound, and distributed at regular intervals. Titles are also listed in the DOCScience Publishing catalogue on the website, refer http://www.doc.govt.nz under Publications, thenScience and Research.
Copyright September 2003, New Zealand Department of Conservation In the interest of forest conservation, DOC Science Publishing supports paperless electronicpublishing. When printing, recycled paper is used wherever possible.
This report was prepared for publication by DOC Science Publishing, Science & Research Unit; editingand layout by Lynette Clelland. Publication was approved by the Manager, Science & Research Unit,Science Technology and Information Services, Department of Conservation, Wellington.
Smit—Marine mammal age and reproductive condition from biopsy sampling EcoRAP, 90 Aeroview Drive, Beach Haven, Auckland, New Zealand Management of marine mammals is often constrained by limited knowledge ofreproductive variables and / or age-specific survival rates. I reviewed the use ofanalyses of tissue samples obtained through biopsy sampling to determinereproductive condition or age of marine mammals, free-ranging cetaceans inparticular. Literature searches show that reproductive condition has beensuccessfully determined from blubber biopsies for one species, while no studyused such biopsies to determine ages of individuals. I suggest that hormonalassays using muscle or blubber tissue obtained from biopsies could easily beused to determine reproductive condition. However, a study needs to beconducted to define age-related changes in selected hormones if hormonalassays of tissues obtained from biopsies are to be used in marine mammal agedetermination.
Keywords: marine mammals, age determination, reproductive condition,biopsy sampling, conservation management.
September 2003, New Zealand Department of Conservation. This paper may be cited as: Smit, A.-M. 2003: Age determination and establishment of reproductive condition from marine mammal biopsy sampling. DOC Science Internal Series 133. Department of Conservation,Wellington. 13 p.
Knowledge of marine mammal life-history and general biology is often limited.
For instance, most of the current understanding of marine mammal physiologyis based on observations of captive individuals (Dierauf & Gulland 2001).
Understanding of free-ranging marine mammals has been limited largely to bio-geographical scales of marine mammal population structures (e.g Baker &Medrano-Gonzalez 2002; Hoelzel 2002). In some instances, particularly with thepinniped and delphinid species, demographic models are commonly used tostudy the dynamics of their populations (Barlow & Boveng 1991).
However, values for demographic variables of marine mammal species, inparticular the cetaceans, are mostly educated guesses because of the difficultyof estimating these variables. For non-marine species, modellers have usedspecies’ populations with good data as surrogates to model another species’population (e.g. Gage & Dyke 1988). Few similar surrogate examples exist formarine mammals, but recently the National Institute of Water and Atmosphere(NIWA) has developed a surrogate population model for Maui’s dolphin(Cephalorhynchus hectori maui) based on life-history knowledge of Hector’sdolphin (Cephalorhynchus hectori hectori) (Sam Ferreira, Department ofConservation, Auckland, pers. comm.). There are likely to be some risksassociated with surrogate approaches to population dynamics. For highlythreatened species, such risks could affect the persistence of the species.
The Department of Conservation (DOC) has a mandate under the MarineMammal Protection Act (1978) to conserve New Zealand’s marine mammalpopulations. This is a daunting task considering that 45 cetacean speciesfrequent New Zealand coastal waters (Baker 1999), of which at least threespecies or sub-species are threatened and endemic. Furthermore, some marinemammal species are taonga for some tangata whenua and are usually highlyvalued by New Zealanders in general. Some marine mammal populations arenow also important economically, forming the basis of a number of tourismventures (e.g. whale watching, swim-with-dolphin tours etc.). Maintainingpopulations of marine mammal species is therefore important from scientific,cultural and aesthetic perspectives.
Normally, management of threatened species focuses on factors affecting oneor two key demographic aspects that are known to result in negative populationgrowth rates. For instance, the establishment of a Marine Mammal Sanctuary atBanks Peninsula along the east coast of the South Island was directed atreducing mortality of both adult and juvenile Hector’s dolphins (Dawson &Slooten 1993). Identifying the most obvious threats to marine mammals is oftenreadily achievable (e.g. Martien et al. 1999), particularly for species with easilymeasurable demographic variables. In general, marine mammal speciesmanagement is constrained by the aspects that are poorly known and notreadily measurable and could benefit from the development of methods todetermine age-specific variables such as survival and reproductive rates.
Recently there has been an increase in genetic studies using samples collectedby biopsy techniques (e.g. Pfeiffer 2002). Following on from this, it has become Smit—Marine mammal age and reproductive condition from biopsy sampling evident that other information could also be gained from these samples. Forinstance, biopsy sampling could be used for regular assessment of disease statusin populations (Kennedy-Stoskopf 2001) or levels of toxic pollution (O’Hara &O’Shea 2001). Considering that demographic models explicitly represent age-structure (Barlow & Boveng 1991) and associated age-specific survival andfecundity schedules, it is likely that the potential exists to use biopsies toestimate such demographic variables for free-ranging marine mammals,cetaceans in particular.
I reviewed the literature on biopsy sampling and associated reproductivecondition and chemical age-determination techniques. I found thatreproductive condition was successfully assessed for one marine mammalspecies using hormonal assays, but could find no report of the age of marinemammals being determined using biopsy sampling. Here I report on myassessment and provide recommendations on the use of biopsy sampling toestimate reproductive condition and age.
I searched eight scientific journals including Marine Mammal Science, Journal ofMammalogy, Journal of Endocrinology, Journal of Molecular Endocrinology,Journal of Reproduction and Fertility, Reproduction, Reviews of Reproductionand Biology of Reproduction as sources for studies on age-determination and /or reproductive condition and focused on papers published since 1995.
I expected that cross-references in manuscripts published in the above journalswould provide further references.
In addition, I was aware that developing techniques are often not reported on injournals early in their development, and therefore conducted an internet searchfor potential additional sources of information. In some instances, my searchesproduced summaries of the interests of researchers that were relevant to myinterests. I contacted some of these people and discussed with them potentialuses of biopsy sampling techniques.
Age determination in marine mammals has been based on counts of growthlayers deposited in persistent tissues such as teeth (e.g. Hohn et al. 1989;Oosthuizen 1997; Pinedo & Hohn 2000) or bone (Klevezal 1996; Marmontel etal. 1996). Body size has also been used as a rough estimate of age (Stevick 1999).
These techniques are all suitable for age determination of dead or captivespecimens, but are difficult or impossible to apply to free-ranging marinemammals, especially cetaceans.
I could find no evidence of age determination using some form of chemicaltechnique that may be applicable to biopsy sampling. Molecular methods todetermine gender are relatively well developed (see Shaw et al. 2003), whilemolecular methods (amino acid racemization) have been used for thedetermination of sample ages of dead tissue, e.g. forensic samples (Ritz-Timmeet al. 1999). As far as I know, no attempt has been made to develop racemizationtechniques applied to biopsy samples to assess age of individual living marinemammals. The influence of aging on DNA polymerase a has been investigated inhumans, but the results were influenced by diet (Srivastava et al. 1999). Wetherefore believe that molecular techniques will be of limited value indeveloping chemically-based age-determination techniques using biopsysamples from living marine mammals.
Hormonal assays have the potential to be developed into age-determinationtechniques. For instance, St Aubin et al. (1996) reported that concentrations ofthyroid and adrenal hormones changed with age in Atlantic bottlenose dolphins(Tursiops truncates). However, St Aubin et al’s (1996) analysis was based onblood samples and the exact relationship between age and hormonalconcentrations has not been defined. One hormone which could beinvestigated as an age-determination method is growth hormone. Favier et al.
(2001) reported that growth hormone concentrations change with age in youngdogs. To date, I know of no studies on marine mammals that have assessedchanges in hormonal concentrations as individuals get older.
R E P R O D U C T I V E C O N D I T I O N Knowledge of cetacean reproduction is limited and is largely based onnecropsies of stranded or killed whales (e.g. Iga et al. 1996), observations ofindividually identified free-ranging animals (e.g. Chapman & Mayo 1987; Steiger& Calambokidis 2000) or tissue samples from captured (e.g. Sawyer-Steffan etal. 1983; Robeck et al. 2001), killed (e.g. Kjeld et al. 1992) or stranded (e.g.
Carballeira et al. 1987) animals. A number of tissues have been used forhormonal studies, but blood has most commonly been used for these (e.g.
Sawyer-Steffan et al. 1983; Schneyer et al. 1985; Kjeld & Olafsson 1987; Kjeld &Theodorsdottir 1991; Kjeld et al. 1992). Reproductive condition has also beenevaluated using analysis of milk (West et al. 2000) and urinary steroids (Walkeret al. 1988; Robeck et al. 1993). For pinnipeds, analyses of saliva and faecalsamples have proven useful for evaluation of steroids (Pietraszek & Atkinson1994).
However, very little use has been made of hormonal assays using biopsysamples collected from live, free-ranging animals. Yoshioka et al. (1994)developed a technique to quantify muscle progesterone that successfullydistinguished between pregnant and non-pregnant minke whales. The muscletechnique is limited, as biopsy dart samples from free-ranging cetaceans oftenconsist of skin and blubber (Brown et al. 1991; Brown et al. 1994), and themuscle-assay would therefore depend on development of a suitable biopsytechnique that targets muscle tissue (Yoshioka et al. 1994).
Smit—Marine mammal age and reproductive condition from biopsy sampling I am aware of one study that used blubber samples to determine pregnancystatus (Mansour et al. 2002). In this study it was shown that progesterone canbe extracted from the blubber of minke whales efficiently enough to allowpregnant and non-pregnant individuals to be identified. Information from thistechnique could be used in the management of a number of cetacean species.
Information on age-distributions and reproductive conditions of free-rangingcetacean populations are limited because of the limited techniques available toestimate these variables. As a result, most cetacean management relies on sometype of interaction model (e.g. Martien et al. 1999), or life-history informationobtained from dead specimens, or incorporates survival and reproductionschedules in a single measure such as population growth rate.
However, cetaceans are primarily threatened through the effect of humanactivities on either survival or reproductive output. For example, commercialwhaling (e.g. Evans 1987), subsistence whaling (e.g. Mitchell & Reeves 1980;Soegiarto & Polunin 1982; Hertz & Kapel 1986) or hunting of small cetaceans(e.g. Smith 1982; Perrin 1985; Price 1985) including live capture fordolphinariums (see Cornell 1984), are all activities that will significantlyinfluence adult survival rate. In some situations smaller cetaceans are hunted orharassed because of their perceived effect on a common food source (e.g.
Miyasaki 1983). Also, incidental catches during commercial fishing operationshave been identified as one of the key factors causing declines in populations ofsome smaller cetacean species (e.g. Smith 1983; Gaskin 1984; Martien et al.
1999). These activities all influence populations primarily through increasingadult mortality.
Human disturbance (e.g. harassment resulting from marine mammal tourism)may have consequences for population dynamics because of effects onbehaviour (e.g. Constantine 1999). This sort of disturbance can change feedingand breeding behaviour, e.g. by affecting reproductive schedules.
Since the 1980s, pollutant levels in cetaceans have been studied and reviewed(Gaskin 1982). The most severe effects of pollution may have consequences foradult survival, but at lower levels the most significant impacts may be onreproductive outputs.
Establishing ways to measure survival and reproductive schedules for free-ranging cetaceans may therefore greatly aid managers in pinpointing keyaspects, such as harassment or pollution, affecting the persistence of species.
My review suggests that reproductive condition of individual marine mammalscould be determined through hormonal assays of tissue (blubber or muscle)samples obtained using biopsy sampling techniques. This could allowproductivity to be estimated for at least a broad adult age-class in a population.
Reproductive condition information can also be used to determine age-classesof marine mammals. Note, however, that sample collection needs to beappropriate, as the assay may be restricted by the type of tissue sampled(Yoshioka et al. 1994).
In contrast to the successful determination and potential determination ofreproductive condition, age determination of live, free-ranging cetaceans hasnever been attempted using biopsy sampling techniques. However, someassociations of hormonal concentrations with age in terrestrial mammalssuggest that assays of selected hormones present in tissue or blubber samplesmay have potential as an aging technique for marine mammals. At present, thelack of understanding of age-related changes in hormonal concentrations inmarine mammals is the prime limitation to development of such a technique.
I believe that biopsy sampling can produce a snapshot of the demography of apopulation by providing information on age distribution, sex ratios, fecundityand survival. This could be useful to managers as it could easily contribute to aLeslie-Matrix or individual-based probability model predicting growth rates andvariables in populations that are most sensitive to changes in environmentalconditions.
It may be possible to use hormonal assays of tissue samples collected throughbiopsies to determine age of free-ranging marine mammals. I am aware ofmuscle and blubber hormonal assay techniques to determine pregnancy statusin free-ranging cetaceans. I suggest that, as a first step to using hormonal assaysto determine age: 1. A theoretical framework be developed for the likely hormonal changes from 2. A study be initiated to determine the potential relationship between these hormonal concentrations and age of an individual. The study should have atwo-pronged approach: a. Collection of tissue biopsies from known-age captive individuals (cetaceans in particular) across the world.
b. Collection of tissue biopsies from stranded individuals in association with collection of teeth or bone samples to use in traditional aging techniques.
3. A study be initiated to develop hormonal assays (both muscle and blubber) from biopsies for a variety of species. This study should also have a two-pronged approach: a. Collection of biopsy samples from captive individuals (particularly cetaceans) where the reproductive status is known across the world.
b. Collection of biopsy samples from stranded individuals in association with autopsy and necropsy results on reproductive status.
Smit—Marine mammal age and reproductive condition from biopsy sampling This report addressed a request for scientific advice from AucklandConservancy and was funded by the Science Advice Fund of DOC (investigationno. 3660. I would like to thank Sam Ferreira for his assistance.
Baker, A.N. 1999: Whales and dolphins of New Zealand and Australia: an identification guide.
Victoria University Press, Wellington.
Baker, C.S.; Medrano-Gonzalez, L. 2002: Worldwide distribution and diversity of humpback whale mitochondrial DNA lineages. In: Pfeiffer, C.J. (Ed.): Molecular and Cell Biology of MarineMammals. Krieger Publishing Company, Malabar, Florida.
Barlow, J.; Boveng, P. 1991: Modeling age-specific mortality for marine mammal populations.
Marine Mammal Science 7: 50–65.
Brown, M.R.; Corkeron, P.J.; Hale, P.T.; Schultz, K.W.; Bryden, M.M. 1994: Behavioral response of east Australian humpback whales, Megaptera novaengliae, to biopsy sampling. MarineMammal Science 10: 391–400.
Brown, M.W.; Kraus, S.D; Gaskin, D.E. 1991: Reaction of North Atlantic right whales (Eubalaena glacialis) to skin biopsy sampling for genetic and pollutant analysis. Report of theInternational Whaling Commision (Special Issue) 13: 81–89.
Carballeira, A.; Brown, J.W.; Fishman, L.M.; Trujillo, D.; Odell, D.K. 1987: The adrenal gland of stranded whales (Kogia breviceps and Mesoplodon europaeus): Morphology, hormonalcontents, and biosynthesis of corticosteroids. General and Comparative Endocrinology68: 293–303.
Constantine, R. 1999: Effects of tourism on marine mammals in New Zealand. Science for Conservation 106, Department of Conservation, Wellington, New Zealand, 60 p.
Cornell, L.H. 1984: Census of captive marine mammals. American Association of Zoological Parks and Aquariums Annual Proceedings: 246–252.
Chapman, P.J.; Mayo, C.A. 1987: Reproduction and recruitment of individually identified humpback whales, Megaptera novaengliae, observed in Massachusetts Bay, 1979–1985. CanadianJournal of Zoology 65: 2853–2863.
Dawson, S.M.; Slooten, E. 1993: Conservation of Hector’s dolphins: The case and process which led to establishment of the Banks Peninsula Marine Mammal Sanctuary. Aquatic Conservation3: 207–221.
Dierauf, L.A.; Gulland, F.M.D. 2001: CRC Handbook of Marine Mammal Medicine. CRC Press LLC, Evans, P.G.H. 1987: The natural history of whales and dolphins. Christofer Helm, London.
Favier, R.P.; Mo, J.A.; Kooistra, H.S.; Rijnberk, A. 2001: Large body size in the dog is associated with transient growth hormone excess at young age. Journal of Endocrinology 170: 479–484.
Gage, T.B.; Dyke, B. 1988: Model life tables for the larger Old World monkeys. American Journal of Primatology 16: 305–320.
Gaskin, D.E. 1982: The ecology of whales and dolphins. Heinemann, London.
Gaskin, D.E. 1984: The harbour porpoise Phocoena phocoena (L.): Regional populations, status, and information on direct and indirect catches. Report of the International WhalingCommission 34: 487–492.
Hertz, O.; Kapel, F.O. 1986: Commercial and subsistence hunting of marine mammals. Ambio 15: Hoelzel, A.R. 2002: Resource specialization and the evolution of population genetic structure in delphinid species. In: Pfeiffer, C.J. (Ed.): Molecular and Cell Biology of Marine Mammals.
Krieger Publishing Company, Malabar, Florida.
Hohn, A.A.; Scott, M.D.; Wells, R.S.; Sweeney, J.C.; Irvine, A.B. 1989: Growth layers in teeth from known age free ranging bottlenose dolphins. Marine Mammal Science 5: 315–342.
Iga, K.; Fukui, Y.; Miyamoto, A.; Ishikawa, H.; Ohsumi, S. 1996: Endocrinological observations of female minke whales (Balaenoptera acutorostrata). Marine Mammal Science 12: 296–301.
Kennedy-Stoskopf, S. 2001: Chapter 15: Viral diseases. In: Dierauf, L.A.; Gulland, F.M.D. (Eds): CRC Handbook of Marine Mammal Medicine. CRC Press LLC, Boca Raton, Florida.
Kjeld, J.M.; Olafsson, I. 1987: Some biochemical parameters in blood and urine of fin whales (Balaenoptera physalus). Israel Journal of Veterinary Medicine 43: 117–121.
Kjeld, J.M.; Sigurjonsson, J.; Arnason, A. 1992: Sex hormone concentrations in blood serum from the North Atlantic fin whale (Baleanoptera physalus). Journal of Endocrinology 134: 405–413.
Kjeld, J.M.; Theodorsdottir, A. 1991: Some electrolytes, hormones and other substances in the blood of fin whales of the coast of Iceland. Natturufroedingurinn 60: 147–154.
Klevezal, G.A 1996: Recording structures of mammals: Determination of age and reconstruction of life history. A.A. Balkema, Rotterdam, Netherlands.
Mansour, A.A.H.; McKay, D.W.; Lien, J.; Orr, J.C.; Banoub, J.H.; Oien, N.; Stenson, G. 2002: Determination of pregnancy status from blubber samples in minke whales (Balaenopteraacutorostrata). Marine Mammal Science 18: 112–120.
Marmontel, M.; O’Shea, T.J.; Kochman, H.; Humphrey, S.R. 1996: Age determination in manatees using growth layer group counts in bone. Marine Mammal Science 12: 54–58.
Martien, K.K.; Taylor, B.L.; Slooten, E.; Dawson, S.M. 1999: A sensitivity analysis to guide research and management for Hector’s dolphin. Biological Conservation 90: 183–191.
Mitchell, E.D.; Reeves, R.R. 1980: The Alaska bowhead problem: a commentary. Arctic 33: 686–723.
Miyasaki, N. 1983: Catch statistics of small cetaceans taken in Japanese waters. Report of the International Whaling Commission 33: 621–631.
O’Hara, T.M.; O’Shea, T.J. 2001: Chapter 22: Toxicology. In: Dierauf, L.A.; Gulland, F.M.D. (Eds): CRC Handbook of Marine Mammal Medicine. CRC Press LLC, Boca Raton, Florida.
Oosthuizen, W.H. 1997: Evaluation of an effective method to estimate age of Cape fur seals using ground tooth sections. Marine Mammal Science 13: 683–693.
Perrin, W.F. 1985: The former dolphin fishery at St. Helena. Report of the International Whaling Commission 35: 423–428.
Pfeiffer, C.J. 2002: Molecular and Cell Biology of Marine Mammals. Krieger Publishing Company, Pietraszek, J.J.; Atkinson, S. 1994: Concentrations of estrone sulphate and progesterone in plasma and saliva, vaginal cytology, and bioelectric impedance during the estrous cycle of theHawaiian monk seal (Monachus schauinslandi). Marine Mammal Science 10: 430–441.
Pinedo, M.C.; Hohn, A.A. 2000: Growth layer patterns in teeth from the fransciscana, Pontoporia blainvillei: developing a model for precision in age estimation. Marine Mammal Science16: 1–27.
Price, W.S. 1985: Whaling in the Caribbean: historical perspective and update. Report of the International Whaling Commission 35: 413–420.
Smit—Marine mammal age and reproductive condition from biopsy sampling Ritz-Timme, S.; Schutz, H.W.; Waite, E.R.; Collins, M.J. 1999: ‘Improvement’ of age estimation using amino acid racemization in a case of pink teeth. American Journal of Forensic Medicine andPathology 20: 216–217.
Robeck, T.R.; Atkinson, S.K.C.; Brook, F. 2001: Chapter 11: Reproduction. In: Dierauf, L.A.; Gulland, F.M.D. (Eds): CRC Handbook of Marine Mammal Medicine. CRC Press LLC, Boca Raton,Florida.
Robeck, T.R.; Schneyer, A.L.; McBain, J.F.; Dalton, L.M.; Walsh, M.T.; Czekala, N.M.; Kraemer, D.C.
1993: Analysis of urinary immunoreactive steroid metabolites and gonadotropins forcharacterization of the estrous cycle, breeding period, and seasonal estrous activity ofcaptive killer whales (Orcinus orca). Zoo Biology 12: 173–187.
Sawyer-Steffan, J.E.; Kirby, V.L.; Gilmartin, W.G. 1983: Progesterone and estrogens in the pregnant and nonpregnant dolphin, Tursiops truncatus, and the effects of induced ovulation. Biologyof Reproduction 28: 897–901.
Schneyer, A.; Castro, A.; Odell, D. 1985: Radioimmunoassay of serum follicle-stimulating hormone and luteinizing hormone in the bottlenosed dolphin. Biology of Reproduction 33: 844–853.
Shaw, C.N.; Wilson, P.J.; White, B.N. 2003: A reliable molecular method of gender determination for mammals. Journal of Mammalogy 84: 123–128.
Smith, T.D. 1982: Current understanding of the status of small cetacean populations in the Black Sea.
FAO Fisheries Series 4: 121–130.
Smith, T.D. 1983: Changes in size of three dolphin (Stenella spp.) populations in the eastern tropical Pacific. Fisheries Bulletin, U.S. 81: 1–13.
Soegiarto, A.; Polunin, N. 1982: The Marine Environment in Indonesia. University of Cambridge/ Srivastava, V.K.; Miller, S.D.; Busbee, D.L. 1999: Aging and DNA polymerase a: modulation by dietary restriction. Journal of Nutrition, Health & Aging 3: 111–120.
St Aubin, D.J.; Ridgway, S.H.; Wells, R.S.; Rhinehart, H. 1996: Dolphin thyroid and adrenal hormones: Circulating levels in wild and semidomesticated Tursiops truncatus, andinfluence of sex, age, and season. Marine Mammal Science 12: 1–13.
Steiger, G.H.; Calambokidis, J. 2000: Reproductive rates of humpback whales off California. Marine Mammal Science 16: 220–239.
Stevick, P.T. 1999: Age-length relationships in humpback whales: a comparison of strandings in the western North Atlantic with commercial catches. Marine Mammal Science 15: 725–737.
Walker, L.A.; Cornell, L.; Dahl, K.D.; Czekala, N.M.; Dargen, C.M.; Joseph, B.; Hsueh, A.J.W.; Lasley, B.L. 1988: Urinary concentrations of ovarian steroid hormone metabolites and bioactivefollicle-stimulating hormone in killer whale (Orcinus orca) during ovarian cycles andpregnancy. Biology of Reproduction 39: 1013–1020.
West, K.L.; Atkinson, S.; Carmichael, M.J.; Sweeney, J.C.; Krames, B.; Krames, J. 2000: Concentrations of progesterone in milk from bottlenose dolphins during differentreproductive states. General and Comparative Endocrinology 117: 216–224.
Yoshiola, M.; Okumura, T.; Aida, K.; Fujise, Y. 1994: A proposed technique for quantifying muscle progesterone content in minke whales (Balaenoptera acutorostrata). Canadian Journalof Zoology 72: 368–370.

Source: http://www.doc.org.nz/documents/science-and-technical/dsis133.pdf