Journal of Chemical Ecology, Vol. 15, No. 8, 1989
ODOR OF THE MUSKOX A Preliminary Investigation
PETER
F. F L O O D , 1 S U Z A N N E and JANICE
R. A B R A M S , 2 G I L L I A N
D. M U I R , t
E. R O W E L L I
~Department of Veterinary Anatomy, Western College of Veterinary Medicine University of Saskatchewan Saskatoon, Saskatchewan, Canada, S7N OWO 2National Research Council Plant Biotechnology Institute 110 Gymnasium Road, Saskatoon, Saskatchewan, Canada, S7N OW9 (Received June 23, 1988; accepted December 8, 1988) Abstract--The behavior of captive male muskoxen was observed closely during their characteristic superiority display, the anatomy of the preputial region was studied in two adults and three calves, and preputial washings and preorbital gland secretion were subjected to gas chromatography and mass spectroscopy. During the superiority display, the prepuce was everted to form a pendulous tube tipped with a fringe of matted hair. Owing to the movement of the animal, the urine that dribbled from the preputial opening was liberally applied to the long guard hairs of the belly. The superiority display was almost exclusively confined to dominant males and apparently accounted for their odor. In the quiescent state, the hair seen around the preputial opening was drawn inside and formed an 8 cm-wide band on the lining of the prepuce. The preputial washings contained large amounts of benzoic acid and p-cresol. The infraorbital gland secretion contained cholesterol, benzaldehyde, and a homologous series of saturated 3,-lactones ranging from 8 to 12 carbons. The latter compounds and the natural secretion smell similar to the human nose. Key Words--Muskox, Ovibos moschatus, odor, preorbital gland, prepuce, rot, urine, behavior, gas chromatography, mass spectroscopy, lactone.
INTRODUCTION T h e m u s k o x h a s t w o p a r t i c u l a r l y n o t i c e a b l e o d o r s , t h e s t r o n g , r a n k s m e l l o f the r u t t i n g m a l e , to w h i c h t h e s p e c i e s p r o b a b l y o w e s its n a m e ( A l l e n , 1913), a n d the light, sweetish, ethereal smell of the preorbital gland secretion. The origin 2207 0098-0331/89/0800-2207506.00/09 1989PlenumPublishingCorporation
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of the rutting odor has never been fully explained, although Teal (1959) recognized that it was associated with urine and was most noticeable in dominant bulls. An analysis of smegma (Teal, 1961, cited by Tener, 1965) indicated that cinnamaldehyde might be an important constituent. The preorbital glands are well developed in muskoxen (Brinkman, 1911, cited by Schaffer, 1940; L6nnberg, 1900; Sack and Ballantyne, 1965), and they are used extensively for self-marking and scent marking in agonistic situations; this occurs during encounters between males and in both sexes when they are alarmed by predators or human activity (Gray, 1987; Gray et al., 1988). A preliminary analysis of preorbital gland tissue from a male muskox revealed the presence of cholesterol, cholesterol esters, and triglycerides but no volatile components were identified (M. Benn, footnote in Gray, 1987). The muskox lacks discrete scent glands in other parts of the body (Schaffer, 1940), although apocrine sweat glands are widely distributed (Flood et al., 1988). There has been some confusion about the existence of interdigital glands in muskoxen (Haltenorth, 1963); our observations (unpublished) confirm L6nnberg's (1900) opinion that they are absent. The object of the present study was to determine the source of the rutting odor and to make a preliminary investigation of the volatiles associated with the preorbital gland and the preputial region.
METHODS AND MATERIALS
Animals. The animals used in the investigation were from the research herd of muskoxen at the Western College of Veterinary Medicine, University of Saskatchewan. The herd originated from 13 hand-reared calves that were captured on Banks Island, Northwest Territories, at about 2 weeks of age in 1982 (Flood et al., 1984). At the time of sample collection, it consisted of six 3year-old females, four entire males (two 3-year-olds and two juveniles) and four 3-year-old castrates. They were kept in a series of interconnecting pens totalling 1.5 hectares. They had free access to brome/alfalfa hay and water and were given 1.5 kg of supplemented oat pellets daily. The animals were thoroughly accustomed to human contact and could be handled without showing signs of distress. General Observations. Day-to-day work with the research herd took 2-10 hr daily and provided a good opportunity for behavioral study. Relevant observations are reported in the results. We also report pertinent information on the anatomy and histology of the preputial region obtained from two adults and three calves that either died or were killed for various reasons. Sample Collection from Preorbital Gland. Preorbital gland secretion was collected by placing pledgets of fine glass wool over the gland openings and
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repeatedly squeezing and wiping the area. The glass wool was cleaned with methylene dichloride (CHzCla) before use and the fingers were wiped with CHzC12 before handling the glass wool. On June 27, secretion was collected from the entire herd and combined to form a single pooled sample. Sample Collection from Prepuce. Preputial odorants were collected by instilling 60 ml of distilled water into the preputial cavity with a syringe, closing the preputial opening with finger and thumb, and massaging the preputial region for a few seconds. The contents of the preputial cavity were then collected in a wide-mouthed glass jar; this sometimes required further massaging of the prepuce. Washes were collected from all the males on June 27 and combined to provide a single sample, which was stored at - 2 0 ~ until analyzed. Extraction of Preorbital Gland Secretion. The glass wool pledgets were extracted with redistilled CH2C12 (BDH, Omnisolve, glass distilled, 3 x 125 ml). The combined extracts were evaporated in a rotary evaporator at 30~ and the residue redissolved in 1 ml CH2C12 prior to gas chromatography. Extraction of Preputial Washes. Following acidification (pH 1, 10% HC1), the washes were extracted separately with redistilled CH2C12 (3 x 50 ml). The extract was dried (anhydrous sodium sulfate) and filtered, then treated as above. Analysis. The preorbital and preputial extracts were treated in the same way. Gas chromatography was performed on a Varian 3700 instrument with a split injector and flame ionization detector. A DB5 column (30 m x 0.32 mm ID) was used with helium as the carrier gas at a flow rate of 85 cm3/sec. The samples were introduced at 50~ then the oven temperature was increased at 5~ to 200~ Mass spectra were obtained on a Finnigan 4000 instrument linked to a model 2300 Incos Data Acquisition system. A DB5 column (60 m • 0.32 mm ID) was used with a splitless injector and helium as the carrier gas at a flow rate of 45 cm3/sec. The oven temperature was raised rapidly from 40~ to 90~ then at 4~ to 300~ An electron impact of 70 eV was used scanning from mass 43 to mass 600 every second. Chemical ionization spectra were also obtained using methane as the reactant gas.
RESULTS
General. The characteristic superiority display known as "head-tilting" (Gray, 1987) was seen when the males entered their first rutting season: a bull showing this behavior walks slowly past his rival with his head tilted to display the horn boss; in doing so he uses a curious stiff-legged gait that emphasizes the height of the shoulder. Close observation revealed that during this process the prepuce was everted to form a pendulous tube about 12 cm in length tipped with a fringe of matted hair (Figure 1). The preputial tube swung about during the display and urine dribbled from its opening. As a result, the abdominal
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portion of the skirt (the long, flowing, outer hairs that give the animal its characteristic appearance) became soaked with urine. This was clearly responsible for the rutting odor. In the quiescent state the preputial tube was retracted to the level of the abdominal wall as in other ruminants and the hair previously visible at its tip was drawn inside. Dissection of the prepuce of a 3.5-year-old bull and a 4-yearold castrate showed that about 8 cm of the prepuce was lined with fine, crimped hair that was up to 3 cm in length. It covered the entire circumference of the preputial cavity and was encrusted with pale caseous particles (Figure 2). Histological examination of the prepuce of three calves less than 2 weeks old showed that the hairs were present at that age and that their follicles were directed proximally in the more distal part of the prepuce and distally in the deeper part. Sebacous glands were associated with the hair follicles, but no sweat glands could be found (Figure 3). Analysis of Preorbital Gland Secretion. Analysis of the methylene chloride extract of the pooled sample by gas chromatography-mass spectrometry allowed
FIG. 1. An adult male muskox with the preputial tube extended and the tuft of hair exposed at its tip. The coat has been trimmed in the area within the circle so that the prepuce can be seen. The inset shows the extended preputial tube in detail. (Composite drawing made from several photographs.)
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FIG. 2. The preputial cavity of a 3.5-year-old muskox opened longitudinally from the ventral side. The tip of the penis is visible on the right and the preputial opening is on the left. The hair-covered zone of the preputial lining is visible between the arrows. (Scale bar = 5 cm.)
FIG. 3. Longitudinal section of the preputial skin in the haired zone from a 1-week-old calf. The lumen of the prepuce is at the top. Parts of hair shafts and several follicles are visible and these are associated with sebaceous glands. Hematoxylin and eosin. (Scale bar = 100 ~m.)
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positive identification of a number of components of the mixture. Assignments of structures were made on the basis of the mass spectral fragmentation patterns observed in both the electron impact and chemical ionization modes, as well as by coelution of authentic standards on gas chromatography. Figure 4 shows the reconstructed ion chromatogram of the preorbital extract. Cholesterol and benzaldehyde were prominent components of the mixture together with a homologous series of straight-chain, saturated 3,-lactones ranging from C8H1402 to C12H2202. The 10-carbon lactone was present in the greatest concentration. The lactones were readily identified in the electron impact mass spectrum by a common lactone fragment (base peak m/z 85), which resulted from cleavage at the ring. The chain lengths were obtained from the chemical ionization experiments and the structural assignments were confirmed by coinjection with commercially available (Aldrich) eight- and 10-carbon 3'lactones. There was a minor peak that eluted ahead of the 12-carbon lactone that also gave a base peak of m/z 85 and a molecular ion at 196 corresponding to a monounsaturated 3,-lactone. Analysis of Preputial Washes. The major components of the CHzC12 extract of the pooled preputial washes were benzoic acid and p-cresol. These assignments were confirmed by nuclear mass resonance. A series of saturated straightchain hydrocarbons ranging from C22H46to C32H66 was present, the C24 homolog being the most prominent. In addition, there was a series of unidentified compounds with longer retention times than cholesterol and with molecular weights and fragmentation patterns similar to those of steroids.
DISCUSSION
Rutting Odor. Self-marking with urine is common in ruminants during the rut. Urine may be applied directly to the neck and beard (goats; Coblentz, 1976), belly (red deer; Lincoln, 1971), or legs (blacktailed deer; Mfiller-Schwarze et al., 1978), or it may be applied indirectly as in the bison (McHugh, 1958) and moose (Geist, 1963), which wallow in urine voided onto the ground, and the camel, which urinates on its tail, then uses this to soak the hind quarters (Wemmer and Murtaugh, 1980). The method of urine self-marking employed by the muskox is apparently unique, and the presence of hair on the inner surface of the prepuce, which is equally unusual, seems to be an anatomical correlate of the urine sprinkling behavior. In the female muskox, a homologous patch of hair is present on the ventral aspect of the vestibule. Teal's (1959) view that the strong rutting odor is most obvious in the dominant male is supported by our own observations on the captive animals in Saskatoon. The superiority display which, among other things, leads to the application of urine to the belly hair is also largely a prerogative of the dominant
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FIG. 4. The reconstructed ion chromatogram of an extract of pooled preorbital gland secretion. The numbered peaks have been identified as follows: (1) benzaldehyde, (2) nonenol, (3) decanol, (4) C8H1402, (5) C9H1602, (6) CioHtsO2, (7) Cl~H2002, (8) a monounsaturated ,/-lactone, (9) C12H2202, (10) myristic acid, (11) C!6H34, (12) Ci9H40, (13) palmitic acid, (14) C20H42, (15) C2LH44, (16) oleic acid, (17) stearic acid, (18) C22H46, (19) C23H48, (20) C24H50, (21) C25H52, (22) phthalate, (23) C26H54, (24) C27H56, (25) C~9H60, (26) cholesterol. The compounds shown by their formulas are straight-chain, saturated hydrocarbons or straight-chain, saturated 7-1actones. The peaks without numbers have not been identified.
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FLooD ET AL.
male (Gray et al., 1988). It therefore seems likely that the characteristic odor of the dominant animal is due to his behavior rather than any profound metabolic change. Nonetheless, such a metabolic change cannot be excluded since changes in testosterone concentration are known to affect the volatile constituents of mammalian urine (Raymer et al., 1986; Schwende et al., 1986), and testosterone levels can influence and be influenced by social status (Bouissou, 1983). Further, it is known that mice (Jones and Nowell, 1974; Lombardi and Vandenberg, 1977), rats (Krames et al., 1969), and rabbits (Bell, 1984) respond differently to the urine of dominant and subordinate conspecifics. The function of the rutting odor is unclear but a number of possibilities exist. It may advertise the presence of a dominant male and discourage interlopers. It may convey the identity of the dominant male to bulls that have challenged him previously and thereby reduce the number of fights. It may suppress reproductive activity in subordinate males (Perret and Schilling, 1987), permitting them to conserve energy. It may advance the onset of reproductive activity in the females of the herd, as does the fleece odor of the ram (Knight and Lynch, 1980) or the voice of the red deer stag (McComb, 1987), allowing the dominant male to minimize the risk that he will be deposed before the females of the herd are ready to mate. Subordinate males may be discouraged from mating with estrous females that already carry the odor of the dominant male. The presence of phenolic compounds in the preputial washings of bull muskoxen is not unexpected since 0.5 % of the content of the preputial gland of the musk deer is p-cresol (Sokolov et al., 1987). The preputial diverticulum of the boar is rich inp-cresol and also contains phenol, o- and m-methoxyphenol and p-ethylphenol (Patterson, 1967), and the urine-soaked belly hair of the rutting red deer contains phenol, p-cresol, and ethylphenol (Albone, 1984). We do not contend that the compounds identified fully account for the rutting odor of the male; clearly, some of the most volatile constituents would have been lost during concentration of the extracts and significant odorants remain unidentified. These considerations apply equally to the preorbital gland secretion considered below. Volatiles were sought in preputial washings as opposed to urine because the odor is likely to be altered by bacterial action in the prepuce; we also believed it was important to collect some of the solids adherent to the preputial hairs. Further, the animals tolerate the procedure well; urine collection on the other hand requires prolonged restraint which causes serious distress, particularly in rutting males. Preorbital Secretion. Perhaps the most characteristic behavior pattern of wild muskoxen is the self-marking sequence in which the preorbital region is rubbed on the outstretched foreleg (Gray, 1987; Gray et al., 1988). This results in the application of secretion to the hair of the leg and face and the release of the volatile constituents into the atmosphere. The preorbital gland may also be
MUSKOXODOR
2215
rubbed on prominent objects in the environment, but this is less frequent. Use of the preorbital gland is associated with agonistic situations, but the nature of the encoded message is unknown. As in the case of rutting odor, many possibilities exist; it may betray individual identity like the inguinal glands of rabbits (Hesterman et al., 1984), and it may convey information about sex and reproductive status. The preorbital glands are much larger in males than females (Gray et al., 1988) and contain apocrine sweat glands and sebaceous components, both of which are influenced by gonadal hormones (Flood, 1985). The presence of large amounts of cholesterol in the preorbital gland secretion is not surprising since this compound is widespread in cutaneous products; straight-chain hydrocarbons are less common, however (Albone, 1984). Benzaldehyde does not seem to be commonly identified in cutaneous secretions, but it is present in urine (Jorgenson et al., 1978) and vaginal secretion (Huggins and Preti, 1976). The 7-1actones found in the preorbital secretion of the muskox are particularly interesting because related compounds are known to be behaviorally active in the black-tailed deer (Mtiller-Schwarze et al., 1978). The pure compounds also smell somewhat similar to the preorbital gland secretion. A compound that is probably isomeric with the "deer lactone," (Z)-4-hydroxy-6-dodecenoic acid lactone (Miiller-Schwarze et al., 1978), was also found, although it was present in smaller amounts than the saturated lactones. 3,-Lactones have been found in the occipital gland of the bactrian camel (Ayorinde et al., 1982), the pedal gland of the bontebok (Burger et al., 1977), and on human hair (Labows et al., 1979). We have made no attempt to determine the absolute stereochemistry of the muskox lactones. Acknowledgments--The Research Herd at the Western College of Veterinary Medicine, Saskatoon, was established with the generous help of the Donner Canadian Foundation and is currently supported by NSERC grants A2759 and A2793. G.D.M. was the recipient of an NSERC Undergraduate Summer Research Award. We would like to thank Colleen Stevens for her dedicated care of the animals, Angela Shaw for expert technical assistance with the chemical analysis, Lawrence Hogge and Doug Olson for the mass spectra, and Juliane Deubner for the artwork.
REFERENCES ALBONE, E.S. 1984. Mammalian Semiochemistry, John Wiley & Sons, Chichester. ALLEN, J.A. 1913. Ontogenic and other variations in muskoxen, with a systematic review of the muskox group, recent and extinct. Memoirs of the American Museum of Natural History 1 : 103226. AYORINDE, F., WHEELER, J.W., WEMMER, C., and MURTAUGH, J. 1982. Volatile components of the occipital gland secretion of the bactrian camel, Camelus bactrianus. J. Chem. Ecol. 8:177183. BELL, D.J. 1984. The rabbits and hares: Order Lagomorpha, pp. 507-530, in R.E. Brown and D.W. Macdonald (eds.). Social Odours in Mammals. Clarendon Press, Oxford.
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BouIssou, M.F. 1983. Androgens, aggressive behaviour and social relationships in higher mammals. Horm. Res. 18:43-61. BURGER, B.V., LEROUX, M., GARBERS,C.F., SPIES, H.S.C., BmALKE,R.C., PACHLER,K.G.R., WESSELS, P.L., CHRIST, V., and MAURER, K.H. 1977. Studies on mammalian pheromones. II. Further compounds from the pedal gland of the bontebok, Damaliscus dorcas dorcas. Z. Naturforsch. 32C:49-56. COBLENTZ, B.E. 1976. Functions of scent-urination in ungulates with special reference to feral goats (Capra hircus). Am. Nat. 110:549-557. FLOOD, P.F. 1985. Sources of significant smells: The skin and other organs, pp. 19-36, in R.E. Brown and D.W. Macdonald (eds.). Social Odours in Mammals. Clarendon Press, Oxford. FLOOD, P.F., ROWELL,J.E., GLOVER,G.J., CHAPLIN,R.K., and HUMPHREYS,S. 1984. The establishment of a small research herd of captive muskoxen, pp. 170-172, in D.R. Klein, R.G. White, and S. Keller (eds.). Proceedings of the First International Muskox Symp., 22-25 May 1983, Fairbanks, Alaska. Biological Papers of the University of Alaska. Special Report No. 4. FLOOD, P.F., STALKER,M.J., and ROWELL, J.E. 1989. The hair follicles and seasonal shedding cycle of the muskox (Ovibos moschatus). Can. J. Zool. 67:1143-1147. GEIST, V. 1963. On the behaviour of the North American moose (Alces alces andersoni Peterson 1950) in British Columbia. Behaviour 20:377-416. GRAY, D.R. 1987. The muskoxen of Polar Bear Pass. National Museum of Natural Sciences/ Fitzhenry and Whiteside, Toronto, 192 pp. GRAY, D.R., FLOOD, P.F., and ROWELL, J.E. 1989. The structure and function of muskox preorbital glands. Can. J. Zool. 67:1134-1142. HALTENORTH,T. 1963. Klassifikation der S~ugetiere: Artiodactyla. Handb. Zool. 8(1): 1-167. HESTERMAN, E.C., MALAFANT,K., and MYKYTOWYCZ,R. 1984. Misidentification by wild rabbits, Oryctolagus cuniculus, of group members carrying the odor of foreign inguinal gland secretion. III. Experiments with mixed sex groups and analysis of further data from all-male and all-female groups. J. Chem. Ecol. 10:403-419. HUGGINS, G.R., and PRETI, G. 1976. Volatile constituents of human vaginal secretions. Am. J. Obstet. Gynecol. 126:129-136. JONES, K., and NOWELL, N. 1974. A comparison of aversive and female attractant properties of urine from dominant and subordinate male mice. Anim. Learn. Behav. 2:141-144. JORGENSON, J.W., NOVOTNY, M., CARMACK,M., COPLAND, G.B., WILSON', S.R., KATONA, S., and WmTTEN, W.K. 1978. Chemical scent constituents in the urine of the red fox (Vulpes vulpes) during the winter season. Science 199:796-798. KRAMES,L., CARR,W.J., and BERGMAN,B. 1969. A pheromone associated with social dominance in male rats. Psychonom. Sci. 16:11-12. KNIGHT, T.W., and LYNCH, P.R. 1980. Source of ram pheromones that stimulate ovulation in the ewe. Anim. Reprod. Sci. 3:133-136. LABOWS,J.N., McGINLEY, K.J., LEYDEN,J.J., and WEBSTER,G.F. 1979. Characteristic 7-lactone odor production of the genus Pityrosporum. Appl. Environ. Microbiol. 38:412-415. LINCOLN, G.A. 1971. The seasonal reproductive changes in the red deer stag (Cervus elaphus). J. Zool., London 163:105-123. LOMBARDI,J.R., and VANDENBERG,J.G. 1977. Pheromonally induced sexual maturation in females: Regulation by the social environment of the male. Science 196:545-546. LONNBERG, E. 1900, On the soft anatomy of the musk-ox (Ovibos moschatus). Proc. Zool. Soc. London 1900:142-167. MCCOMB, K. 1987. Roaring by red deer stags advances the date of oestms in hinds. Nature 330:648649. MCHUGH, T. 1958. Social behavior of the American buffalo (Bison bison bison). Zoologica, NY 43:1-40. MOLLER-SCHWARZE,D., RAVID, U., CLAESSON,A., SINGER, A.E., SILVERSTEIN,R.M., MOLLER-
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SCHWARZE, C., VOLKMAN,N.J., ZEMANEK,K.F., and BUTLER, R.G. 1978. The deer-lactone: Source, chemical properties and responses of black-tailed deer. J. Chem. Ecol. 4:247-256. PATTERSON, R.L.S. 1967. A possible contribution of phenolic components to boar odour. J. Sci. Food Agric. 18:8-10. PERRET, M., and SCHmHNG, A. 1987. Intermale sexual effect elicited by volatile urinary ether extract in Microcebus murinus (Prosimian, Primates). J. Chem. Ecol. 13:495-507. RAYMER,J., WIESLER, D., NOVOTNY, M., ASA, C., SEAL, U.S., and MECH, L.D. 1986. Chemical scent constituents in urine of wolf (Canis lupus) and their dependence on reproductive hormones. J. Chem. Ecol. 12:297-314. SACK, W.O., and BALLANTYNE, J.H. 1965. Anatomical observations on a muskox calf (Ovibos moschatus) with particular reference to thoracic and abdominal topography. Can. J. Zool. 43:1033-1047. SCHAFFER, J. 1940. Die Hautdrtisenorgane der S~iugetiere. Urban and Schwarzenberg, Berlin. SCHWENDE, F.T., WIESLER, D., JORGENSON,J.W., CARMACK,M., and NOVOTNY, M. 1986. Urinary volatile constituents of the house mouse, Mus musculus, and their endocrine dependency. J. Chem. Ecol. 12:277-296. SOKOLOV, V.E., KAGAN, M.Z., VASILIEVA,V.S., PRIHODKO, X.X, and ZINKEVICH, E.P. 1987. Musk deer (Moschus moschiferus): reinvestigation of the main lipid components from preputial gland secretion. J. Chem. Ecol. 13:71-83. TEAL, J.J., JR. 1959. Muskox in rut. Polar ]Votes 1:65-71. TENER, J.S. 1965. Muskoxen in Canada, a biological and taxonomic review. Canadian Wildlife Service. Monograph No. 2 Queen's Printer, Ottawa. 166 pp. WEMMER, C., and MURTAU~H, J. 1980. Olfactory aspects of rotting behaviour in the bactrian camel, Camelus bactrianusferus, pp. 107-124, in D. Mfiller-Schwarze and R.M. Sitverstein (eds.). Chemical Signals: Vertebrates and Aquatic Invertebrates. Plenum, New York.
ODOR OF THE MUSKOX A Preliminary Investigation
PETER
F. F L O O D , 1 S U Z A N N E and JANICE
R. A B R A M S , 2 G I L L I A N
D. M U I R , t
E. R O W E L L I
~Department of Veterinary Anatomy, Western College of Veterinary Medicine University of Saskatchewan Saskatoon, Saskatchewan, Canada, S7N OWO 2National Research Council Plant Biotechnology Institute 110 Gymnasium Road, Saskatoon, Saskatchewan, Canada, S7N OW9 (Received June 23, 1988; accepted December 8, 1988) Abstract--The behavior of captive male muskoxen was observed closely during their characteristic superiority display, the anatomy of the preputial region was studied in two adults and three calves, and preputial washings and preorbital gland secretion were subjected to gas chromatography and mass spectroscopy. During the superiority display, the prepuce was everted to form a pendulous tube tipped with a fringe of matted hair. Owing to the movement of the animal, the urine that dribbled from the preputial opening was liberally applied to the long guard hairs of the belly. The superiority display was almost exclusively confined to dominant males and apparently accounted for their odor. In the quiescent state, the hair seen around the preputial opening was drawn inside and formed an 8 cm-wide band on the lining of the prepuce. The preputial washings contained large amounts of benzoic acid and p-cresol. The infraorbital gland secretion contained cholesterol, benzaldehyde, and a homologous series of saturated 3,-lactones ranging from 8 to 12 carbons. The latter compounds and the natural secretion smell similar to the human nose. Key Words--Muskox, Ovibos moschatus, odor, preorbital gland, prepuce, rot, urine, behavior, gas chromatography, mass spectroscopy, lactone.
INTRODUCTION T h e m u s k o x h a s t w o p a r t i c u l a r l y n o t i c e a b l e o d o r s , t h e s t r o n g , r a n k s m e l l o f the r u t t i n g m a l e , to w h i c h t h e s p e c i e s p r o b a b l y o w e s its n a m e ( A l l e n , 1913), a n d the light, sweetish, ethereal smell of the preorbital gland secretion. The origin 2207 0098-0331/89/0800-2207506.00/09 1989PlenumPublishingCorporation
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FLOODET AL.
of the rutting odor has never been fully explained, although Teal (1959) recognized that it was associated with urine and was most noticeable in dominant bulls. An analysis of smegma (Teal, 1961, cited by Tener, 1965) indicated that cinnamaldehyde might be an important constituent. The preorbital glands are well developed in muskoxen (Brinkman, 1911, cited by Schaffer, 1940; L6nnberg, 1900; Sack and Ballantyne, 1965), and they are used extensively for self-marking and scent marking in agonistic situations; this occurs during encounters between males and in both sexes when they are alarmed by predators or human activity (Gray, 1987; Gray et al., 1988). A preliminary analysis of preorbital gland tissue from a male muskox revealed the presence of cholesterol, cholesterol esters, and triglycerides but no volatile components were identified (M. Benn, footnote in Gray, 1987). The muskox lacks discrete scent glands in other parts of the body (Schaffer, 1940), although apocrine sweat glands are widely distributed (Flood et al., 1988). There has been some confusion about the existence of interdigital glands in muskoxen (Haltenorth, 1963); our observations (unpublished) confirm L6nnberg's (1900) opinion that they are absent. The object of the present study was to determine the source of the rutting odor and to make a preliminary investigation of the volatiles associated with the preorbital gland and the preputial region.
METHODS AND MATERIALS
Animals. The animals used in the investigation were from the research herd of muskoxen at the Western College of Veterinary Medicine, University of Saskatchewan. The herd originated from 13 hand-reared calves that were captured on Banks Island, Northwest Territories, at about 2 weeks of age in 1982 (Flood et al., 1984). At the time of sample collection, it consisted of six 3year-old females, four entire males (two 3-year-olds and two juveniles) and four 3-year-old castrates. They were kept in a series of interconnecting pens totalling 1.5 hectares. They had free access to brome/alfalfa hay and water and were given 1.5 kg of supplemented oat pellets daily. The animals were thoroughly accustomed to human contact and could be handled without showing signs of distress. General Observations. Day-to-day work with the research herd took 2-10 hr daily and provided a good opportunity for behavioral study. Relevant observations are reported in the results. We also report pertinent information on the anatomy and histology of the preputial region obtained from two adults and three calves that either died or were killed for various reasons. Sample Collection from Preorbital Gland. Preorbital gland secretion was collected by placing pledgets of fine glass wool over the gland openings and
MUSKOX ODOR
2209
repeatedly squeezing and wiping the area. The glass wool was cleaned with methylene dichloride (CHzCla) before use and the fingers were wiped with CHzC12 before handling the glass wool. On June 27, secretion was collected from the entire herd and combined to form a single pooled sample. Sample Collection from Prepuce. Preputial odorants were collected by instilling 60 ml of distilled water into the preputial cavity with a syringe, closing the preputial opening with finger and thumb, and massaging the preputial region for a few seconds. The contents of the preputial cavity were then collected in a wide-mouthed glass jar; this sometimes required further massaging of the prepuce. Washes were collected from all the males on June 27 and combined to provide a single sample, which was stored at - 2 0 ~ until analyzed. Extraction of Preorbital Gland Secretion. The glass wool pledgets were extracted with redistilled CH2C12 (BDH, Omnisolve, glass distilled, 3 x 125 ml). The combined extracts were evaporated in a rotary evaporator at 30~ and the residue redissolved in 1 ml CH2C12 prior to gas chromatography. Extraction of Preputial Washes. Following acidification (pH 1, 10% HC1), the washes were extracted separately with redistilled CH2C12 (3 x 50 ml). The extract was dried (anhydrous sodium sulfate) and filtered, then treated as above. Analysis. The preorbital and preputial extracts were treated in the same way. Gas chromatography was performed on a Varian 3700 instrument with a split injector and flame ionization detector. A DB5 column (30 m x 0.32 mm ID) was used with helium as the carrier gas at a flow rate of 85 cm3/sec. The samples were introduced at 50~ then the oven temperature was increased at 5~ to 200~ Mass spectra were obtained on a Finnigan 4000 instrument linked to a model 2300 Incos Data Acquisition system. A DB5 column (60 m • 0.32 mm ID) was used with a splitless injector and helium as the carrier gas at a flow rate of 45 cm3/sec. The oven temperature was raised rapidly from 40~ to 90~ then at 4~ to 300~ An electron impact of 70 eV was used scanning from mass 43 to mass 600 every second. Chemical ionization spectra were also obtained using methane as the reactant gas.
RESULTS
General. The characteristic superiority display known as "head-tilting" (Gray, 1987) was seen when the males entered their first rutting season: a bull showing this behavior walks slowly past his rival with his head tilted to display the horn boss; in doing so he uses a curious stiff-legged gait that emphasizes the height of the shoulder. Close observation revealed that during this process the prepuce was everted to form a pendulous tube about 12 cm in length tipped with a fringe of matted hair (Figure 1). The preputial tube swung about during the display and urine dribbled from its opening. As a result, the abdominal
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FLOOD ET AL.
portion of the skirt (the long, flowing, outer hairs that give the animal its characteristic appearance) became soaked with urine. This was clearly responsible for the rutting odor. In the quiescent state the preputial tube was retracted to the level of the abdominal wall as in other ruminants and the hair previously visible at its tip was drawn inside. Dissection of the prepuce of a 3.5-year-old bull and a 4-yearold castrate showed that about 8 cm of the prepuce was lined with fine, crimped hair that was up to 3 cm in length. It covered the entire circumference of the preputial cavity and was encrusted with pale caseous particles (Figure 2). Histological examination of the prepuce of three calves less than 2 weeks old showed that the hairs were present at that age and that their follicles were directed proximally in the more distal part of the prepuce and distally in the deeper part. Sebacous glands were associated with the hair follicles, but no sweat glands could be found (Figure 3). Analysis of Preorbital Gland Secretion. Analysis of the methylene chloride extract of the pooled sample by gas chromatography-mass spectrometry allowed
FIG. 1. An adult male muskox with the preputial tube extended and the tuft of hair exposed at its tip. The coat has been trimmed in the area within the circle so that the prepuce can be seen. The inset shows the extended preputial tube in detail. (Composite drawing made from several photographs.)
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FIG. 2. The preputial cavity of a 3.5-year-old muskox opened longitudinally from the ventral side. The tip of the penis is visible on the right and the preputial opening is on the left. The hair-covered zone of the preputial lining is visible between the arrows. (Scale bar = 5 cm.)
FIG. 3. Longitudinal section of the preputial skin in the haired zone from a 1-week-old calf. The lumen of the prepuce is at the top. Parts of hair shafts and several follicles are visible and these are associated with sebaceous glands. Hematoxylin and eosin. (Scale bar = 100 ~m.)
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positive identification of a number of components of the mixture. Assignments of structures were made on the basis of the mass spectral fragmentation patterns observed in both the electron impact and chemical ionization modes, as well as by coelution of authentic standards on gas chromatography. Figure 4 shows the reconstructed ion chromatogram of the preorbital extract. Cholesterol and benzaldehyde were prominent components of the mixture together with a homologous series of straight-chain, saturated 3,-lactones ranging from C8H1402 to C12H2202. The 10-carbon lactone was present in the greatest concentration. The lactones were readily identified in the electron impact mass spectrum by a common lactone fragment (base peak m/z 85), which resulted from cleavage at the ring. The chain lengths were obtained from the chemical ionization experiments and the structural assignments were confirmed by coinjection with commercially available (Aldrich) eight- and 10-carbon 3'lactones. There was a minor peak that eluted ahead of the 12-carbon lactone that also gave a base peak of m/z 85 and a molecular ion at 196 corresponding to a monounsaturated 3,-lactone. Analysis of Preputial Washes. The major components of the CHzC12 extract of the pooled preputial washes were benzoic acid and p-cresol. These assignments were confirmed by nuclear mass resonance. A series of saturated straightchain hydrocarbons ranging from C22H46to C32H66 was present, the C24 homolog being the most prominent. In addition, there was a series of unidentified compounds with longer retention times than cholesterol and with molecular weights and fragmentation patterns similar to those of steroids.
DISCUSSION
Rutting Odor. Self-marking with urine is common in ruminants during the rut. Urine may be applied directly to the neck and beard (goats; Coblentz, 1976), belly (red deer; Lincoln, 1971), or legs (blacktailed deer; Mfiller-Schwarze et al., 1978), or it may be applied indirectly as in the bison (McHugh, 1958) and moose (Geist, 1963), which wallow in urine voided onto the ground, and the camel, which urinates on its tail, then uses this to soak the hind quarters (Wemmer and Murtaugh, 1980). The method of urine self-marking employed by the muskox is apparently unique, and the presence of hair on the inner surface of the prepuce, which is equally unusual, seems to be an anatomical correlate of the urine sprinkling behavior. In the female muskox, a homologous patch of hair is present on the ventral aspect of the vestibule. Teal's (1959) view that the strong rutting odor is most obvious in the dominant male is supported by our own observations on the captive animals in Saskatoon. The superiority display which, among other things, leads to the application of urine to the belly hair is also largely a prerogative of the dominant
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FIG. 4. The reconstructed ion chromatogram of an extract of pooled preorbital gland secretion. The numbered peaks have been identified as follows: (1) benzaldehyde, (2) nonenol, (3) decanol, (4) C8H1402, (5) C9H1602, (6) CioHtsO2, (7) Cl~H2002, (8) a monounsaturated ,/-lactone, (9) C12H2202, (10) myristic acid, (11) C!6H34, (12) Ci9H40, (13) palmitic acid, (14) C20H42, (15) C2LH44, (16) oleic acid, (17) stearic acid, (18) C22H46, (19) C23H48, (20) C24H50, (21) C25H52, (22) phthalate, (23) C26H54, (24) C27H56, (25) C~9H60, (26) cholesterol. The compounds shown by their formulas are straight-chain, saturated hydrocarbons or straight-chain, saturated 7-1actones. The peaks without numbers have not been identified.
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2214
FLooD ET AL.
male (Gray et al., 1988). It therefore seems likely that the characteristic odor of the dominant animal is due to his behavior rather than any profound metabolic change. Nonetheless, such a metabolic change cannot be excluded since changes in testosterone concentration are known to affect the volatile constituents of mammalian urine (Raymer et al., 1986; Schwende et al., 1986), and testosterone levels can influence and be influenced by social status (Bouissou, 1983). Further, it is known that mice (Jones and Nowell, 1974; Lombardi and Vandenberg, 1977), rats (Krames et al., 1969), and rabbits (Bell, 1984) respond differently to the urine of dominant and subordinate conspecifics. The function of the rutting odor is unclear but a number of possibilities exist. It may advertise the presence of a dominant male and discourage interlopers. It may convey the identity of the dominant male to bulls that have challenged him previously and thereby reduce the number of fights. It may suppress reproductive activity in subordinate males (Perret and Schilling, 1987), permitting them to conserve energy. It may advance the onset of reproductive activity in the females of the herd, as does the fleece odor of the ram (Knight and Lynch, 1980) or the voice of the red deer stag (McComb, 1987), allowing the dominant male to minimize the risk that he will be deposed before the females of the herd are ready to mate. Subordinate males may be discouraged from mating with estrous females that already carry the odor of the dominant male. The presence of phenolic compounds in the preputial washings of bull muskoxen is not unexpected since 0.5 % of the content of the preputial gland of the musk deer is p-cresol (Sokolov et al., 1987). The preputial diverticulum of the boar is rich inp-cresol and also contains phenol, o- and m-methoxyphenol and p-ethylphenol (Patterson, 1967), and the urine-soaked belly hair of the rutting red deer contains phenol, p-cresol, and ethylphenol (Albone, 1984). We do not contend that the compounds identified fully account for the rutting odor of the male; clearly, some of the most volatile constituents would have been lost during concentration of the extracts and significant odorants remain unidentified. These considerations apply equally to the preorbital gland secretion considered below. Volatiles were sought in preputial washings as opposed to urine because the odor is likely to be altered by bacterial action in the prepuce; we also believed it was important to collect some of the solids adherent to the preputial hairs. Further, the animals tolerate the procedure well; urine collection on the other hand requires prolonged restraint which causes serious distress, particularly in rutting males. Preorbital Secretion. Perhaps the most characteristic behavior pattern of wild muskoxen is the self-marking sequence in which the preorbital region is rubbed on the outstretched foreleg (Gray, 1987; Gray et al., 1988). This results in the application of secretion to the hair of the leg and face and the release of the volatile constituents into the atmosphere. The preorbital gland may also be
MUSKOXODOR
2215
rubbed on prominent objects in the environment, but this is less frequent. Use of the preorbital gland is associated with agonistic situations, but the nature of the encoded message is unknown. As in the case of rutting odor, many possibilities exist; it may betray individual identity like the inguinal glands of rabbits (Hesterman et al., 1984), and it may convey information about sex and reproductive status. The preorbital glands are much larger in males than females (Gray et al., 1988) and contain apocrine sweat glands and sebaceous components, both of which are influenced by gonadal hormones (Flood, 1985). The presence of large amounts of cholesterol in the preorbital gland secretion is not surprising since this compound is widespread in cutaneous products; straight-chain hydrocarbons are less common, however (Albone, 1984). Benzaldehyde does not seem to be commonly identified in cutaneous secretions, but it is present in urine (Jorgenson et al., 1978) and vaginal secretion (Huggins and Preti, 1976). The 7-1actones found in the preorbital secretion of the muskox are particularly interesting because related compounds are known to be behaviorally active in the black-tailed deer (Mtiller-Schwarze et al., 1978). The pure compounds also smell somewhat similar to the preorbital gland secretion. A compound that is probably isomeric with the "deer lactone," (Z)-4-hydroxy-6-dodecenoic acid lactone (Miiller-Schwarze et al., 1978), was also found, although it was present in smaller amounts than the saturated lactones. 3,-Lactones have been found in the occipital gland of the bactrian camel (Ayorinde et al., 1982), the pedal gland of the bontebok (Burger et al., 1977), and on human hair (Labows et al., 1979). We have made no attempt to determine the absolute stereochemistry of the muskox lactones. Acknowledgments--The Research Herd at the Western College of Veterinary Medicine, Saskatoon, was established with the generous help of the Donner Canadian Foundation and is currently supported by NSERC grants A2759 and A2793. G.D.M. was the recipient of an NSERC Undergraduate Summer Research Award. We would like to thank Colleen Stevens for her dedicated care of the animals, Angela Shaw for expert technical assistance with the chemical analysis, Lawrence Hogge and Doug Olson for the mass spectra, and Juliane Deubner for the artwork.
REFERENCES ALBONE, E.S. 1984. Mammalian Semiochemistry, John Wiley & Sons, Chichester. ALLEN, J.A. 1913. Ontogenic and other variations in muskoxen, with a systematic review of the muskox group, recent and extinct. Memoirs of the American Museum of Natural History 1 : 103226. AYORINDE, F., WHEELER, J.W., WEMMER, C., and MURTAUGH, J. 1982. Volatile components of the occipital gland secretion of the bactrian camel, Camelus bactrianus. J. Chem. Ecol. 8:177183. BELL, D.J. 1984. The rabbits and hares: Order Lagomorpha, pp. 507-530, in R.E. Brown and D.W. Macdonald (eds.). Social Odours in Mammals. Clarendon Press, Oxford.
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BouIssou, M.F. 1983. Androgens, aggressive behaviour and social relationships in higher mammals. Horm. Res. 18:43-61. BURGER, B.V., LEROUX, M., GARBERS,C.F., SPIES, H.S.C., BmALKE,R.C., PACHLER,K.G.R., WESSELS, P.L., CHRIST, V., and MAURER, K.H. 1977. Studies on mammalian pheromones. II. Further compounds from the pedal gland of the bontebok, Damaliscus dorcas dorcas. Z. Naturforsch. 32C:49-56. COBLENTZ, B.E. 1976. Functions of scent-urination in ungulates with special reference to feral goats (Capra hircus). Am. Nat. 110:549-557. FLOOD, P.F. 1985. Sources of significant smells: The skin and other organs, pp. 19-36, in R.E. Brown and D.W. Macdonald (eds.). Social Odours in Mammals. Clarendon Press, Oxford. FLOOD, P.F., ROWELL,J.E., GLOVER,G.J., CHAPLIN,R.K., and HUMPHREYS,S. 1984. The establishment of a small research herd of captive muskoxen, pp. 170-172, in D.R. Klein, R.G. White, and S. Keller (eds.). Proceedings of the First International Muskox Symp., 22-25 May 1983, Fairbanks, Alaska. Biological Papers of the University of Alaska. Special Report No. 4. FLOOD, P.F., STALKER,M.J., and ROWELL, J.E. 1989. The hair follicles and seasonal shedding cycle of the muskox (Ovibos moschatus). Can. J. Zool. 67:1143-1147. GEIST, V. 1963. On the behaviour of the North American moose (Alces alces andersoni Peterson 1950) in British Columbia. Behaviour 20:377-416. GRAY, D.R. 1987. The muskoxen of Polar Bear Pass. National Museum of Natural Sciences/ Fitzhenry and Whiteside, Toronto, 192 pp. GRAY, D.R., FLOOD, P.F., and ROWELL, J.E. 1989. The structure and function of muskox preorbital glands. Can. J. Zool. 67:1134-1142. HALTENORTH,T. 1963. Klassifikation der S~ugetiere: Artiodactyla. Handb. Zool. 8(1): 1-167. HESTERMAN, E.C., MALAFANT,K., and MYKYTOWYCZ,R. 1984. Misidentification by wild rabbits, Oryctolagus cuniculus, of group members carrying the odor of foreign inguinal gland secretion. III. Experiments with mixed sex groups and analysis of further data from all-male and all-female groups. J. Chem. Ecol. 10:403-419. HUGGINS, G.R., and PRETI, G. 1976. Volatile constituents of human vaginal secretions. Am. J. Obstet. Gynecol. 126:129-136. JONES, K., and NOWELL, N. 1974. A comparison of aversive and female attractant properties of urine from dominant and subordinate male mice. Anim. Learn. Behav. 2:141-144. JORGENSON, J.W., NOVOTNY, M., CARMACK,M., COPLAND, G.B., WILSON', S.R., KATONA, S., and WmTTEN, W.K. 1978. Chemical scent constituents in the urine of the red fox (Vulpes vulpes) during the winter season. Science 199:796-798. KRAMES,L., CARR,W.J., and BERGMAN,B. 1969. A pheromone associated with social dominance in male rats. Psychonom. Sci. 16:11-12. KNIGHT, T.W., and LYNCH, P.R. 1980. Source of ram pheromones that stimulate ovulation in the ewe. Anim. Reprod. Sci. 3:133-136. LABOWS,J.N., McGINLEY, K.J., LEYDEN,J.J., and WEBSTER,G.F. 1979. Characteristic 7-lactone odor production of the genus Pityrosporum. Appl. Environ. Microbiol. 38:412-415. LINCOLN, G.A. 1971. The seasonal reproductive changes in the red deer stag (Cervus elaphus). J. Zool., London 163:105-123. LOMBARDI,J.R., and VANDENBERG,J.G. 1977. Pheromonally induced sexual maturation in females: Regulation by the social environment of the male. Science 196:545-546. LONNBERG, E. 1900, On the soft anatomy of the musk-ox (Ovibos moschatus). Proc. Zool. Soc. London 1900:142-167. MCCOMB, K. 1987. Roaring by red deer stags advances the date of oestms in hinds. Nature 330:648649. MCHUGH, T. 1958. Social behavior of the American buffalo (Bison bison bison). Zoologica, NY 43:1-40. MOLLER-SCHWARZE,D., RAVID, U., CLAESSON,A., SINGER, A.E., SILVERSTEIN,R.M., MOLLER-
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SCHWARZE, C., VOLKMAN,N.J., ZEMANEK,K.F., and BUTLER, R.G. 1978. The deer-lactone: Source, chemical properties and responses of black-tailed deer. J. Chem. Ecol. 4:247-256. PATTERSON, R.L.S. 1967. A possible contribution of phenolic components to boar odour. J. Sci. Food Agric. 18:8-10. PERRET, M., and SCHmHNG, A. 1987. Intermale sexual effect elicited by volatile urinary ether extract in Microcebus murinus (Prosimian, Primates). J. Chem. Ecol. 13:495-507. RAYMER,J., WIESLER, D., NOVOTNY, M., ASA, C., SEAL, U.S., and MECH, L.D. 1986. Chemical scent constituents in urine of wolf (Canis lupus) and their dependence on reproductive hormones. J. Chem. Ecol. 12:297-314. SACK, W.O., and BALLANTYNE, J.H. 1965. Anatomical observations on a muskox calf (Ovibos moschatus) with particular reference to thoracic and abdominal topography. Can. J. Zool. 43:1033-1047. SCHAFFER, J. 1940. Die Hautdrtisenorgane der S~iugetiere. Urban and Schwarzenberg, Berlin. SCHWENDE, F.T., WIESLER, D., JORGENSON,J.W., CARMACK,M., and NOVOTNY, M. 1986. Urinary volatile constituents of the house mouse, Mus musculus, and their endocrine dependency. J. Chem. Ecol. 12:277-296. SOKOLOV, V.E., KAGAN, M.Z., VASILIEVA,V.S., PRIHODKO, X.X, and ZINKEVICH, E.P. 1987. Musk deer (Moschus moschiferus): reinvestigation of the main lipid components from preputial gland secretion. J. Chem. Ecol. 13:71-83. TEAL, J.J., JR. 1959. Muskox in rut. Polar ]Votes 1:65-71. TENER, J.S. 1965. Muskoxen in Canada, a biological and taxonomic review. Canadian Wildlife Service. Monograph No. 2 Queen's Printer, Ottawa. 166 pp. WEMMER, C., and MURTAU~H, J. 1980. Olfactory aspects of rotting behaviour in the bactrian camel, Camelus bactrianusferus, pp. 107-124, in D. Mfiller-Schwarze and R.M. Sitverstein (eds.). Chemical Signals: Vertebrates and Aquatic Invertebrates. Plenum, New York.
