Assessment of Dominance Hierarchy Through Urine Scent Marking and Its Chemical Constituents in Male Blackbuck Antelope Cervicapra, a Critically Endangered Species

Behavioural Processes 85 (2010) 58–67

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Behavioural Processes

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Assessment of dominance hierarchy through urine scent marking and its chemical constituents in male blackbuck Antelope cervicapra, a critically endangered species

Thangavel Rajagopal a,b, Govindaraju Archunan a,∗, Pitchairaj Geraldine c, Chellam Balasundaram c a Center for Pheromone Technology, Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India b Department of Biotechnology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi-626123, Tamil Nadu, India c Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India article info abstract

Article history: In ungulates the process of chemical communication by urinary scent marking has been directly related to Received 18 February 2010 reproductive dominance, territorial defense and proximity to resources. The differences in the frequency Received in revised form 7 May 2010 of urine marking and chemical composition of urine of males Antelope cervicapra before, during and after Accepted 3 June 2010 the dominance hierarchy period were assessed. The variations in the urine marking and its chemical profiles of dominant males (n = 9), bachelors (n = 5) and sub-adult males (n = 5) were compared to find Keywords: out how the dominance hierarchy influences the confined blackbuck herd under semi-natural captive Dominance hierarchy conditions. The frequency of urine marking is significantly higher (p < 0.001) in dominant males. Twenty- Urinary scent marking Indian Blackbuck eight major constituents were identified in the urine of dominant males (before, during and after the Chemical profiles dominance hierarchy period), bachelor and sub-adult males. Among these, three specific compounds Subordinate male namely, 3-hexanone (I), 6-methyl-5-hepten-2-one (II) and 4-methyl-3-heptanone (III) were seen only in dominant males urine during the dominance hierarchy period. Based on the behavioural observation and the unique chemical constituents in the urine, it is concluded that the dominant male scent odor suppresses aggression, scent marking, scent production and territorial patrolling activities of subordinate males, through which the dominant male establish their hierarchy and attains success in reproduction. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Lewis, 2005; Equus Caballus: Kimura, 2001; Meriones unguiculatus: Shimozuru et al., 2006). Many ungulates are socially characterized by well-defined sta- Typically urinary scent marking involves deposition of social ble dominance hierarchies (Cassinello, 1995; Cote, 2000; Freeman pheromones to elicit response from a conspecific (Ewer, 1968; et al., 2004; Roden et al., 2005) which often determines the first Bowyer et al., 1994; Hoffman et al., 2010). Scent marking or the best access to food, social interactions, and choice of mate behaviours of many ungulates have been described (Gosling, 1985; (Roden et al., 2005; Hemelrijik et al., 2008). The hierarchical posi- Bowyer et al., 1994); urine and scent glands are the major sources tion of an individual is influenced by various factors including age of the odors (Roberts and Dunbar, 2000; Gosling and Roberts, 2001; (Bison bison: Maher and Byers, 1987; Robitaille and Prescott, 1993), Lewis, 2005). Major functions of urinary scent marking are defense body weight (Gazella dama: Cassinello and Pieters, 2000), both age of territory and resources, advertisement of social status, regula- and body weight (Bison bison: Roden et al., 2005), aggressiveness tion of social relationships, mate attraction, and advertisement of (Oreamnos americanus: Cote, 2000; Capra hircus: Barroso et al., reproductive condition (Halpin, 1986; Penn and Potts, 1998; Smith 2000; Loxodonta africana: Ganswindt et al., 2005), androgen level et al., 2001; Brennan and Kendrick, 2006). (Pupu puda: Barto et al., 1998; Elaphurus davidianus: Li et al., 2004; In many ungulates urinary scent marking behaviour not only Pan troglodytes: Muller and Wrangham, 2004) and dominance hier- mediates aggressive interactions between males but also facili- archy is characterized by scent marking/production (Lemur catta: tates male–female interactions. For example, odors of urinary scent Kappeler, 1990; Mus domesticus: Hurst, 1990; Oreotragus oreotra- marking help to establish the territory of dominant male and gus: Roberts and Dunbar, 2000; Propithecus uerreauxi uerreauxi: also to keep the other males away from its territory (Thomsons’s gazelles, Gazella Thomson: Estes, 1967; Blackbuck, Antelope cervicapra: David, 1973). In ungulates like bontebok, Damaliscus dorcas ∗ Corresponding author. Tel.: +91 431 2407040; fax: +91 431 2407045. dorcas (Schaller, 1967), springbok, Antidorces marsupialis (David, E-mail address: [email protected] (G. Archunan). 1973) and wildebeest, Connochaetes taurinus (Estes, 1969) urinary

0376-6357/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2010.06.007 T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67 59 scent marking directs other males as part of a ritualized challenge. Table 1 Urinary scent marking in North American elk, Cervus elaphus pri- Antelope cervicapra L.: identification features of adult male blackbucks. marily influences the dominance interactions between adult males Animal (No.) Number of Identification characters and the urinary pheromones might advertise the physical condi- twists in horn tion of males (McCullough, 1969; Bowyer and Kitchen, 1987). Male 1st 4a Narrow horns with pointed tips; urine excreted during rut has a strong, pungent and unique odor A large size mole in the lower abdomen on which relays information an dominance hierarchy to conspecifics left side 2nd 3a Almost parallel horns with pointed tips (Miquelle, 1991) indicating the urine of males may possess critically 3rd 3a Left horn normal; right horn damaged and important components required in establishing territory and/to half bent attract females. 4th 4a Slightly twisted narrow horns with curved Urinary constituents of a few ungulates have been character- tips pointing each other a ized, but the biological role of urinary scent marking is yet to be 5th 3 Broad and compressed horn; tip of the right horn curved outward investigated. For example, the red deer urine consisted mainly 6th 3a Long broad horns with curved tips outward that derivative of carboxylic acids and some aromatic compounds 7th 3a Broad horns with their tips curved inward (Bakke and Figenschou, 1990). Volatile substances identified in 8th 3a Long narrow horns with their tips curved the urine of white-tailed deer comprise alcohol, aldehyde, furan, inward 9th 3a Long broad horn; tip of the right horn ketone, nitrite, alkene, alkane, thiol ester, disulfide, aromatic ether, damaged ketal and amine classes of compounds (Miller et al., 1998). The pre- 10th 2a Narrow twisted horns, tip of the both selection of candidate substances has further resulted in successful horns very long and pointed characterization of a few biological urinary pheromones in vari- 11th 3a Narrow horns, tip of the left horn little ous zoo animals, farm animals and rodents. For example, urine of damaged 12th 3b Long twisted slim horn, tip of the right Asian female elephants in estrus contain high concentration of a horn curved inward; right horn outward volatile (Z)-7-dodecen-1-yl acetate compound which function as 13th 2a Long parallel horns with their tips straight a sex pheromone stimulating male sexual behaviour (Rasmussen onward sky et al., 1997). In addition, the estrus-specific urinary volatile, 1- 14th 2a Broad horns; a large size scare in the right iodoundecane may function as bull attractant (Rameshkumar and hind leg Archunan, 2002). The male mice urinary pheromones like 2-sec- a Thick solid horn. b butyl-dihydrothiazole and dehydro-exo-brevicomin were active in Slim solid horn. eliciting inter-male aggression (Novotny et al., 1985a), attractive- ness to females (Jemiolo et al., 1985), and estrus synchronization (Jemiolo et al., 1986). Another urinary volatile compound 2,5- 2.2. Study animals dimethylpyrazine delivered from grouped adult females, delayed sexual maturation among young female mice (Novotny et al., In the beginning of the study period (June 2007) the Blackbuck 1985b). The urinary compounds and putative pheromones of dom- enclosure contained a total population of 75 animals. The group inant and subordinate male blackbucks are yet to be identified. was classified (Prasad, 1983) into 14 adult males, 21 adult females, The Indian Blackbuck, A. cervicapra is territorial and generally 16 sub-adult males, 18 sub-adult females and 6 young ones. The lives in herds 30–100 individuals. The regulation of its social life social hierarchy system (i.e. dominance hierarchy) was studied depends to a large extent on chemical communication as reflected in 14 adult males only. Each individual was recognized by varia- by its various odoriferous skin glands and other sources of chemical tion in shape of the horn and additional morphological features signals. Each group has a territory within which a linear domi- (Table 1). nance hierarchy is formed by means of aggression interactions. Previous study has demonstrated that the Indian Blackbucks scent 2.3. Social status of male blackbuck mark their territory with urine and preorbital glands (David, 1973; Manimozhi, 1996; Rajagopal and Archunan, 2008; Rajagopal et al., The nomenclature of male social status are (Hogg and Forbes, 2010). We tested the hypothesis that there were differences in the 1997; Mungall, 1977): (1) harem masters (i.e. dominant males) frequency of scent urination and its chemical composition in domi- who hold harems, (2) challengers (i.e. predominant males) with- nant male before, during and after the dominance hierarchy period out their own harems, but challenge the harem master and try to when compared with that of bachelor male. The study is a first hold females, (3) the bachelor group which stay away from female step in understanding the potential role of urine marking in the groups. Indian Blackbucks change their social status from ‘bache- dominance hierarchy of Indian Blackbuck. lor’ to ‘challenger’ and became ‘harem masters’; but some of them (bachelors) may never reach the highest social rank during their lifetime (Mungall, 1977). 2. Material and methods 2.4. Behavioural observation 2.1. Study area Frequency of urine marking behaviour was observed in adult This study was conducted in the conservation and breeding cen- males (n = 14) using focal sampling method (Altmann, 1974). The ◦ ◦ tre of Arignar Anna Zoological Park (AAZP) (13 16 S and 79 54 E observations were made for 122 days during 18 months from June at an altitude of MSL+ 10–100 m), Vandalur, Chennai, South India. 2007 to December 2008. The observation schedule was divided into Chennai has the distinction of being the first zoo in India, started in two shifts: morning 08.00–10.00 h and afternoon 14.00–16.00 h. 1855. In 1976, the zoo was moved to the Vandalur Reserve Forest The duration of each watch for 2 h each comprised, a total of 4 h comprising, an area of about 510 ha near Chennai. The habitat of of observation per day. The animals were directly observed using AAZP is considered a tropical evergreen scrub, a degraded forest 8 × 40 binoculars in front of the enclosure. The observation included mostly consisting of thorny bushes. The average annual rainfall is mapping all visible scent marks and recording the type and loca- ◦ about 250 mm and the temperature about 26 C. tions of marks. 60 T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67

2.5. Urine collection software 11th version to compare the frequency of urine marking before, during, and after the formation of social hierarchy period in Urine samples were collected from dominant males before, dur- the dominant and bachelor males. ing and after the formation of dominance hierarchy period and also from bachelors and sub-adult males for chemical analysis. To 3. Results minimize the effect of individual variation the urine samples were pooled group wise [dominant (n = 9), bachelor (n = 5) and sub-adult 3.1. Duration of dominancy or leadership period males (n = 5)]. Urine was collected from the floor immediately after voiding using a clean, dry 1 cc syringe, and placed in a 2 cc vial. Among the 14 adult male Blackbuck observed under the semi- Once collected, the vials were labelled and placed in a cold ther- natural captive conditions, 9 were found to have established mos flask until reaching the camp at 10.30 am and 5.30 pm, where dominance hierarchy during the study period from June 2007 to the samples were placed in a freezer at −20 ◦C for GC–MS analysis. December 2008. In the beginning the animal number 4 (14th June to 17th July 2007) exhibited dominance and took over the other males 2.6. Extract preparation followed by animal numbers 5, 8, 6, 1, 11, 14, 12 and 10, respectively (Table 2). Animal number 14 showed the longest duration of Dichloromethane (DCM) was used as a solvent in GC–MS analy- leadership in the same enclosure for 210 days from 4th May 2008 sis. From each sample 5 ml was taken and mixed with 5 ml DCM (1:1 to 29th November 2008. Subsequently animal numbers 6, 1, and 11 ratio) and filtered through a silica gel column (60–120 meshes) for took over the leadership same re-emerging as dominant male (ani- 30 min at room temperature. The filtered extract which was 1/5 of mal number 6 exhibited four times; animal number 1 three times its original volume was cooled with liquid nitrogen to condense it. and animal number 11 two times); however, the duration of leadership period of these animals was vary and ranged between 3 and 2.7. GC–MS analysis 20 days. However, animal numbers 2, 3, 7, 9, and 13 never reached higher social ranking (dominant or predominant) during the entire The GC–MS analysis were made using a QP-5000 (Schimadzu, study period. Japan). The 2 ␮l of extract was injected into the GC–MS on a 30 m glass capillary column with a film thickness of 0.25 ␮m 3.2. Urine marking behaviour (30 m × 0.2 mm i.d. coated with UCON HB 2000) using the follow- The frequency of urine marking was recorded in 14 adult males ing temperature programme, initial oven temperature of 40 ◦C for (Fig. 1). The frequency of urine marking was recorded as follows: 4 min increased to 250 ◦C at a rate of 15 ◦C for 10 min. The gas chro- 2.57 ± 0.36 to 4.25 ± 0.18, before the hierarchy formation period; matography (Schimadzu GC 15A) was equipped with FID detector 3.0 ± 0.57 to 5.75 ± 0.27 during the hierarchy formation period; connected to an integrator. The area under each peak was used 1.0 ± 0.00 to 3.2 ± 0.36 after the hierarchy formation period in the for quantitative calculations. The detection accuracy was about dominant males and 0.25 ± 0.25 to 1.65 ± 0.15 in bachelor males 1 ng/peak. The relative amount of each component was reported as (Table 3). The urine marking frequency (F = = 37.47), was the percent of the ion current. The GC–MS was under the computer 0.001;3.59 significantly higher during the formation of hierarchy period fol- control at 70 eV using ammonia as reagent gas at 95 eV performed lowed by before and after the hierarchy period in the dominant chemical ionization. Identification of unknown compounds was males as compared to bachelor males (Tables 4 and 4a). Further, the made by probability-based matching using the computer Library frequency of urine marking and duration of leadership are directly built within the NICT 12 system. (r = 0.816, p < 0.001) related in dominant males (Fig. 2).

2.8. Statistical analysis 3.3. Urinary chemical proﬁles

The total amount of urine marking behaviour of dominant and The proﬁles of volatile compounds obtained before, during and subordinate males was performed using the mean ± SE. The data after the formation of dominance hierarchy periods in the urine of for the frequency of urine marking behaviour was analysed using dominant male (Fig. 3A–C), bachelor (Fig. 4A) and sub-adult male the one-factor analysis of variance (ANOVA) with SPSS statistical (Fig. 4B). Each male sample mix contained 10–26 detectable peaks.

Table 2 Duration of leadership in 9 dominant males from June 2007 to December 2008.

Animal (No.) Duration of leadership Days (No.) Remarks

From To

4 14 June 2007 17 July 2007 35 Natural death due to ageing (12 April 2008) 5 18 July 2007 30 October 2007 105 Natural death (3 December 2008) 8 31 October 2007 12 February 2008 104 Death after leg injury (30 March 2008) 6 12 February 2008 21 February 2008 10 1 21 February 2008 3 March 2008 11 6 3 March 2008 22 March 2008 20 11 23 March 2008 27 March 2008 5 1 28 March 2008 2 April 2008 6 6 3 April 2008 8 April 2008 6 1 8 April 2008 21 April 2008 14 6 21 April 2008 23 April 2008 3a Death due to injury during inﬁghting (23 April 2008) 11 23 April 2008 3 May 2008 12 14 4 May 2008 29 November 2008 210b Death due to eye injury during inﬁghting (17 December 2008) 12 29th November 2008 8th December 2008 11 10 8th December 2008 21st December 2008 14

a Minimum duration of leadership. b Maximum duration of leadership. T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67 61

Table 3 Antelope cervicapra L. frequency (mean ± SE) of urine marking behaviour by adult males (n = 14) during leadership period (days) from June 2007 to December 2008.

Fig. 1. Breeding lek or resting place and associated scent marking behaviour: (A) territorial or dominant male leg scratching display at dung pile or defecation place, (B) snifﬁng display at dung pile, (C) urination display at dung pile and (D) defecation display at dung pile. 62 T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67

Table 4 Antelope cervicapra L. One-way ANOVA with post hoc comparison of urine marking behaviour in dominant male before, during and after hierarchy period as compared to bachelor male.

Variation SS df MS F Sig.

Between groups 82.445 3 27.482 37.476 0.001* Within groups 41.066 56 0.733

Total 123.511 59

The means were compared using DMRT. * Statistically highly signiﬁcant (p < 0.001).

Nearly 28 detectable peaks were noted in the dominant, bachelor and sub-adult males. The peak numbers correspond to the list of compounds identified (Tables 5 and 6). Visual examination of all the chromatograms showed that there was a consistent quali- tative difference in the chemical profiles before, during and after the formation of dominance hierarchy period between dominant, bachelor and sub-adult males. Among the 28 volatile compounds, 22, 26 and 13 compounds were identified in the urine sample of dominant males before, during and after the formation of dominance hierarchy period, respectively. Further, 12 compounds in the urine of bachelor males and 10 compounds in the urine of sub-adult males were identified. Comparison of the identified compounds before, during and after the formation of dominance hierarchy periods of dominant, bachelor and sub-adult males revealed that certain compounds at peak height were reduced or disappeared or specific to a particular period in dominant, bachelor and sub-adult males. For

Fig. 3. GC–MS proﬁles of volatile compounds in the urine of dominant males before (A), during (B) and after (C) the formation of dominance hierarchy period. Peak number refers to Table 4.

instance, among the 26 compounds present in the urine sample of dominant male during the formation of dominance hierarchy period, three compounds viz., 3-hexanone (I), 6-methyl-5-hepten- 2-one (II) and 4-methyl-3-heptanone (III) are speciﬁc during the

Fig. 2. Relationship between duration of leadership period and their relative frequency of urine marking behaviour in dominant males. Linear ﬁt of regression described by the function y = 3.63x + 0.067*. DM: dominant male. Animal number 6, 1, and 11 reappeared as leader (A.No. 6 exhibited four times; A.No. 1 three times and A.No. 11 two times).

Table 4a Comparison of means from Table 4 using DMRT post hoc tests.

Subset for alpha = 0.05

123

Bachelor male 1.0440 After formation of hierarchy 1.6147 Before formation of hierarchy 2.9687 During formation of hierarchy 4.0707 Signiﬁcance 0.087 1.000 1.000

Uses harmonic mean sample size = 15.000. Means within a subset are not different Fig. 4. GC–MS proﬁles of volatile compounds in the urine of bachelor (A) and sub- from each other. adult male blackbucks (B) peak number refers to Table 4. T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67 63

Table 5 Antelope cervicapra L. volatile compounds in the urine of dominant and subordinate male.

Peak No. Compound name Dominant (hierarchy period) Subordinate

Before During After Bachelor Sub-adult

1 2-Propenoic acid +++ +++ +++ +++ −−− 2 2,2-Dimethyl propane +++ +++ −−− −−− −−− 3 3-Hexanone (I) −−− +++ −−− −−− −−− 4 2,2-Dimethyl butane +++ +++ +++ +++ +++ 5 Phenol +++ +++ −−− −−− −−− 6 1,5-Hexadien-3-ol +++ +++ −−− −−− −−− 7 2-Methyl cyclopentanone +++ +++ −−− −−− +++ 8 3-Methyl-1-pentanol +++ +++ +++ +++ +++ 9 6-methyl-5-hepten-2-one (II) −−− +++ −−− −−− −−− 10 2,2-Dimethyl propanoic acid +++ +++ +++ +++ +++ 11 3-Methyl-2-methyl pyridine +++ +++ −−− −−− −−− 12 4-Methyl phenol −−− −−− +++ −−− −−− 13 3-Methyl-2-methyl hexane +++ +++ −−− −−− −−− 14 4-Pyridine carboxylic acid +++ +++ −−− −−− −−− 15 2,2-Dimethyl heptane +++ +++ +++ +++ +++ 16 4-Pentanal +++ +++ +++ +++ +++ 17 4-Methyl-3-haptanone (III) −−− +++ −−− −−− −−− 18 2,2,5,5-Tetramethyl hexane +++ +++ +++ +++ +++ 19 Bis-(1,1-dimethyl ethyl) diazene +++ +++ +++ +++ +++ 20 3,5-Dimethyl-1-hexane +++ +++ −−− −−− −−− 21 1-Bromo-2,2-dimethyl propane +++ +++ +++ +++ +++ 22 1,2-Dimethyl-3-(1-methyl ethyl) cyclopentanol −−− −−− +++ +++ +++ 23 3,7-Dimethyl-1-octanol +++ +++ +++ +++ −−− 24 2-Methyoxyl-2-butanoic acid +++ +++ +++ −−− −−− 25 2,9-Dimethyl decane −−− +++ −−− +++ −−− 26 6-Ethyl-2-methyl decane +++ +++ −−− −−− −−− 27 1-Chloro tetradecane +++ +++ −−− −−− −−− 28 1-Iodo octane +++ +++ −−− −−− −−−

Total number of volatiles 22 26 13 12 10

Abbreviation: +++, present; −−−, absent. formation of dominance hierarchy period. However, the peaks during the hierarchy period than before hierarchy period; but of 10 compounds i.e. 2, 5, 6, 11, 13, 14, 20, 26, 27 and 28 these were not present after the completion of hierarchy period (2,2-dimethyl propane, phenol, 1,5-hexadien-3-ol, 3-methyl-2- in dominant male, also bachelor and sub-adult male urine sam- methyl pyridine, 3-methyl-2-methyl hexane, 2-pyridine carboxylic ple. acid, 3,5-dimethyl-1-hexane, 6-ethyl-2-methyl decane, 1-chloro Further, the compound 2-methyoxyl-2-butanoic acid (Peak tetradecane, 1-ido octane) appeared higher in the dominant male No. 24) appeared before, during and after the formation of

Table 6 Volatile compounds identiﬁed in the urine of dominant and subordinate male and their nature of compound, molecular weight and formula.

Peak no. Compound Nature Molecular weight Molecular formula

1 2-Propenoic acid Carboxylic acid 72 C3H4O2 2 2,2-Dimethyl propane Alkane 72 C5H12 3 3-Hexanone (I) Ketone 100 C6H12O 4 2,2-Dimethyl butane Alkane 86 C6H14 5 Phenol Phenol 94 C6H6O 6 1,5-Hexadien-3-ol Alcohol 98 C6H10O 7 2-Methyl cyclopentanone Ketone 98 C6H10O 8 3-Methyl-1-pentanol Alcohol 102 C6H14O 9 6-Methyl-5-hepten-2-one (II) Ketone 126 C8H14O 10 2,2-Dimethyl propanoic acid Carboxylic acid 102 C5H10O2 11 3-Methyl-2-methyl pyridine Pyridine 107 C7H9N 12 4-Methyl phenol Phenol 108 C7H8O 13 3-Methyl-2-methyl hexane Alkane 114 C8H18 14 4-Pyridine carboxylic acid Carboxylic acid 123 C6H5NO2 15 2,2-Dimethyl heptane Alkane 128 C9H20 16 4-Pentanal Aldehyde 130 C8H18O 17 4-Methyl-3-haptanone (III) Ketone 166 C10H14O2 18 2,2,5,5-Tetramethyl hexane Alkane 142 C10H22 19 Bis-(1,1-dimethyl ethyl) diazene Alkene 142 C8H18N 20 3,5-Dimethyl-1-hexane Alkane 145 C10H25 21 1-Bromo-2,2-dimethyl propane Alkane 151 C5H11Br 22 1,2-Dimethyl-3-(1-methyl ethyl) cyclopentanol Alcohol 156 C10H20O 23 3,7-Dimethyl-1-octanol Alcohol 158 C10H22O 24 2-Methyoxyl-2-butanoic acid Carboxylic acid 162 C8H18O3 25 2,9-Dimethyl decane Alkane 170 C12H26 26 6-Ethyl-2-methyl decane Alkane 184 C13H28 27 1-Chloro tetradecane Alkane 232 C14H29Cl 28 1-Iodo octane Alkane 240 C8H17I 64 T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67

Fig. 5. Distribution of dung pile (i.e. places of urination and defecation) in a Blackbuck enclosure. * Represents scattered dung pile of dominant male in and around food trough in the enclosure. Dung piles of all bachelor blackbucks are scattered only near edge of the enclosure. dominance hierarchy periods only in the dominant male urine. It is interesting to note that in the present study the domi- The compounds present in all male urine samples, were 2,2- nant male often preferred particular place for their urine marking dimethyl butane, 3-methyl-1-pentanol, 2,2-dimethyl propanoic close to the food trough (i.e. focal point of his territory or breeding acid, 2,2-dimethyl heptane, 4-pentanal, 2,2,5,5-tetramethyl hex- lek) (Fig. 5). A similar result was obtained by several workers who ane, bis-(1,1-dimethyl ethyl) diazene and 1-bromo-2,2-dimethyl found that the male ungulates commonly chose the place for the propane (i.e. during the three different stages in dominant, bachelor urination in order to make the territorial determination (Gosling, and sub-adult males). 1985; Azeve do et al., 1996). It indicates the dominant male gen- All the compounds identified in the urine of dominant, bachelor erally uses urine as a reliable signal for territory ownership and and sub-adult male, had a molecular weight above 72 and less than as a warning at the border to other males within the territorial 240. The gas chromatography analysis showed that the compounds region. It is also reported that the occurrence of male urine mark- fall between the retention times between 5 and 45 min. ing is related to female reproductive state (Converse et al., 1995; Gould and Overdorff, 2002). Therefore, the present results suggest that urine marks may play a major role in the defense of terri- 4. Discussion tories against potential intruders and to advertise their agonistic dominance over the other males and or to attract the females. The frequency of urine marking by the dominant male during Reappearance (i.e. repeated occurrence) of leader and the for- hierarchy formation period (p < 0.001) is higher when compared mation of hierarchy were observed in the animal numbers 6, 1 and to before and after the hierarchy period as compared to bache- 11 during February to April 2008 though for a short period (A.No. lor males. The result is in agreement with earlier investigations, 6: 20–3 days; A.No. 1: 14–6 days; A.No. 11: 12–5 days). In mature which have described that the dominant male urine scent mark- free-ranging bulls the dominance relationship between males were ing behaviour (i.e. odor) may adversely affect the fighting ability, transitory and unstable (Lott, 1979). In our study, though during rut suppressing the social and sexual behaviour of subordinate males periods (August–October and March–May) the males loose hierar- in several species like pine vole, Microtus pinetoram (Brant et al., chy (i.e. rank) it was re-established subsequently. Rajagopal (2009) 1998), wolves, Canis lupus (Derix et al., 1993), dwarf monogooses, reported that more females exhibited heat during these periods. Helagale parvula (Clutton-Brock et al., 2001) and African wild dogs, Therefore, the present results clearly indicate that the formation Lycaon pictus (Courchamp et al., 2002). Primates like Saguinus and maintenance of dominance hierarchy system is mandatory for oedipus (French et al., 1984), S. fuscicollis (Epple and Eatz, 1984) breeding purpose. and Callithrix kuhli (Smith et al., 1997) the dominant male primer In the present study, twenty-eight urinary volatile compounds pheromones create hormonal suppression; the dominant females were identified in the dominant, bachelor and sub-adult males. scent marking odors inhibit the reproductive cyclicity in subor- The volatiles identified in the urine belong to the alkanes, alkenes, dinate females (Smith and Abbott, 1998; Wirtu et al., 2004). The alcohols, ketones, phenols, pyridine and carboxylic acids classes higher frequency of urine marking behaviour is directly associated of compounds. These classes of compounds have already been with higher levels of gonadal (i.e. testosterone) steroids (Thiessen reported from the urine of other mammals (Dominic, 1991; Miller and Rice, 1976; Arteaga et al., 2008) and facilitates attraction of et al., 1998; Rekwot et al., 2001; Archunan, 2009). opposite sex (Novotny et al., 1990; Rich and Hurst, 1998, 1999) and Urinary compounds identified as unique to dominant male dur- advertise the ownership and competitive ability (Thom and Hurst, ing dominance hierarchy period had molecular weight of <300 2004). and had fewer than 20 carbon atoms. Airborne pheromones usu- T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67 65

Table 7 Pheromones reported in various animals and their functions.

S. No. Compound Animal Function Reference

1 3-Hexanonea White-tailed deer Higher concentration during breeding season Miller et al. (1998) as compared to non-breeding season 2 (Z)-4-Hepten-2-oneb Novel moth Female attracting Ekpa et al. (1985) 3 6-Methyl-6-hepten-2-oneb Black beetles Aggregation Zhu et al. (1994) 4 (E)-4-Methyl-4-hepten-3-oneb Palpatore Defensive Ken and Kenji (2001) 5 6-Methyl-5-hepten-2-onea Ant Alarm Moser et al. (1968) 6 6-Hydroxy-6-methyl-3-heptanoneb Mice Accelerate puberty Novotny et al. (1999) 7 5-Hydroxy-4-methyl-3-heptanoneb Rice weevil Aggregation Walgenbach et al. (1987) 8 2-Heptanoneb Honeybee Attractant in wasp Raymer et al. (1986) 9 3-Hexanone Blackbuck To be elucidated This publication 10 6-Methyl-5-hepten-2-one Blackbuck To be elucidated This publication 11 4-Methyl-3-haptanone Blackbuck To be elucidated This publication

a Compounds similar to our identified compounds. b Compounds with slight variation of structure our identified compounds. ally contain 5–20 carbon atoms and must be volatile to reach the chy period may provide behaviourally important chemical cues receiver; pheromones typically have molecular weight about less suggesting their role in aggression to male and attraction to than 300 (Bradbury and Vehrencamp, 1998). For example, in whit- female. tailed deer, several volatile compounds occur exclusively in the It is well established that urine is a primary medium for urine of dominant males during the mating season that had molec- excreting metabolized hormone i.e. testosterone, it may play ular weight <300 (Miller et al., 1998). Similarly, in red deer, volatile a role in the degree of variability in urinary volatiles and aid compounds possess molecular weight <300 (Bakke and Figenschou, in individual recognition (Novotny et al., 1985a,b; Rasmussen 1990). Female Asian elephant release a urinary pheromone with 13 et al., 1997). Many of the by-products of the action of these carbons and a molecular weight near 300 and proved to attract male steroids, as well as the excreted steroid metabolites, are reflected (Rasmussen et al., 1997). In the cow, a specific volatile occur exclu- in the urinary volatiles. Interestingly, the 10 urinary volatile sively in urine during the heat period that had molecular weight (2,2-dimethyl propane, phenol, 1,5-hexadien-3-ol, 3-methyl-2- less than 300 (Rameshkumar et al., 2000). Therefore, the dominant methyl pyridine, 3-methyl-2-methyl hexane, 4-pyridine carboxylic unique urinary compounds have physical properties necessary for acid, 3,5-dimethyl-1-hexane, 6-ethyl-2-methyl decane, 1-chloro consideration as putative pheromones. tetradecane and 1-ido octane) compounds appeared before and The majority of identified urinary compounds are long chain during the hierarchy formation period in dominant male, but dis- alkanes. Long chain hydrocarbons are commonly encountered in appeared after the hierarchy period (Table 5). This is reflected in the both plant and animal kingdom. In fact n-alkanes are among changes of their particular constituents after the hierarchy forma- the commonest constituents of plant wax and play a major role tion period in dominant male, while these compounds could have in bitrophic herbivore-plant interactions (Udayagiri and Marson, some role in chemo signals of the endocrine status. It is reported 1997). In a few mammals they play a significant role in sex- that the compounds 2,3-dehydro-exo brevicomin (Novotny et al., ual attraction (Dominic, 1991; Archunan, 2009). For example, an 1984) and 3-ethyl-2,7-dimethyl octane (Achiraman and Archunan, alkane, 1,5-diemethyl-6-8-dioxodicyclo (3,2,1) octane acts as a 2005) are found to be unique urinary constituents of the intact pheromone during musth in elephants (Rasmussen and Perrin, male mouse as compared to castrated mouse, however, these com- 1999). Before and during hierarchy period the compounds may pounds reappeared following testosterone treatment in castrated be act as chemical signals are 2,2-dimethyl propane, 3-methyl-2- male. Hence, the result provides support to the concept that certain methyl hexane, 3,5-dimethyl-1-hexane, 6-ethyl-2-methyl decane, urinary volatile compounds may influence the hormonal as well as 1-chloro tetradecane and 1-iodo octane may be the putative chem- social status. ical signals appearing prior to dominant hierarchy period. It is The present study documents for the first time the chemical important to note that the peak area and peak height of these constituents in the urine of male Indian Blackbucks. Significant compounds 3,5-dimethyl-1-hexane, 6-ethyl-2-methyl decane, 1- variations in the frequency of urine marking behaviour and chem- chloro tetradecane and 1-iodo octane in the GC profile were ical composition were observed in the dominant males before, comparatively higher during hierarchy period than in the before during and after the hierarchy period when compared with that hierarchy period. This suggests that these compounds may be of bachelor males. Based on the behavioural evidence and the role maximally produced during hierarchy period even though their of volatile compounds in the urine supported by literature, it is production is inhibited after the hierarchy period of the dominant concluded that the dominant male may use urine for territorial male. marking, to attract opposite sex and to deter the subordinates to In this study in dominant male urine 26 volatile compounds maintain the social hierarchy. Additional behavioural research on have been identified during the hierarchy formation period. Among the functional role of the assessed compounds is needed to deter- them, three are volatile compounds, 3-hexanone (I), 6-methyl-5- mine the role of different compounds in social communication. hepten-2-one (II) and 4-methyl-3-haptanone (III) in the molecular weight range between 84 and 166 and having C6 to C10 car- Acknowledgements bon atoms. Of these compounds, compound (I) has also been reported in the white-tailed deer dominant male urine during The authors thank Prof. B.V. Burger, Department of Chemistry, breeding season (Miller et al., 1998). Interestingly the compound University of Stellenbosh, South Africa, for encouragement and (II) has been reported as pheromones in the novel moth, black suggestion, Prof. P.Govindarajalu, for critically sign through the beetles, palpatore, ant and mice; the compound (III) has also manuscript and the Chief Wildlife Warden and the Director of been already reported as urinary pheromone in the female mice, the AAZP, Vandalur, Chennai, for granting permission to carry rice weevil and honeybee (Table 7). Hence, the appearance of out this study. The authors are indebted to Dr K. Senthilkumar these specific compounds in dominant male urine during hierar- (Veterinary Assistant Surgeon) and Dr Pathan Nazrulla Khan (Zoo 66 T. Rajagopal et al. / Behavioural Processes 85 (2010) 58–67

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