IJEB 56(11) 781-794.Pdf

Indian Journal of Experimental Biology Vol. 56, November 2018, pp. 781-794

Inter-relationship of behaviour, faecal testosterone levels and glandular volatiles in determination of dominance in male Blackbuck

Thangavel Rajagopal1,2, Ponnirul Ponmanickam3, Arunachalam Chinnathambi4, Parasuraman Padmanabhan5, Balazs Gulyas5 & Govindaraju Archunan1*

1Pheromone Technology Lab, Department of Animal Science, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India 2Department of Zoology and Microbiology, Thiagarajar College (Autonomous), Madurai-625 009, Tamil Nadu, India 3Department of Zoology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi 626 124, Tamil Nadu, India 4Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia-11451 5Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore-637553 Received 28 March 2017; revised 25 November 2017

The social hierarchy of blackbuck plays a crucial role in mate selection and establishment of hierarchy in order to maintain successful reproduction. The factors that influence dominancy have not been yet investigated in the Indian male blackbuck. Here, we investigated the interrelationships between behaviours (aggressive and scent marking), chemical profiles of preorbital gland secretion and faecal testosterone levels in male blackbuck with special reference to dominance hierarchy. The frequency of aggressive behaviour, preorbital gland scent marking behaviour and faecal testosterone level were significantly higher (P <0.001) in the dominant males than the other males. Among the 43 major volatile compounds identified in the pre-orbital gland posting of dominant and subordinate male Blackbucks, four compounds viz., 2-methyl propanoic acid (I), 2-methyl-4-heptanone (II), 2,7-dimethyl-1-octanol (III) and 1,15-pentadecanediol (IV) were present only in the preorbital gland post of the dominant male during the hierarchy period. The results suggest that male blackbuck preorbital gland marking behaviour is meant to defend the home range or territorial region, and the scent odour suppresses aggression, scent marking, scent production and territorial patrolling of subordinate males. The behavioural expression, level of testosterone and volatile chemical profiles are closely interlinked towards establishment of dominance in the blackbuck. This investigation is the first of its kind to detect establishment of dominance hierarchy based on identification of volatile chemical compounds of preorbital gland and assessment of faecal testosterone in blackbuck.

Keywords: Antelope cervicapra (L), Dominance hierarchy, Faecal testosterone, Indian antelope, Preorbital gland secretion, Scent marking behaviour

Social dominance is a central issue in studies of marking from dominant males is far more effective in animal behaviour, and of great interest to behavioural promoting puberty acceleration in young female mice biologists. Dominance hierarchies often determine the than urine marking from subordinate males4. Since the first or the best access to food, social interaction, or social subordination suppresses gonadal function, mating within the animal groups1. The hierarchial emission of dominant male-specific chemical signals position of given individual can be influenced by its (pheromones) is under testosterone control. In age, body weight, aggressiveness, endocrine status addition, the subordinate males occupy less desirable i.e., androgen production, and/or scent habitats leading to lesser survival rate as well as lower marking/production2. Male dominance status is not reproductive potential4. simply a function of aggressiveness, but acquisition Scent marking by mammals is thought to be and maintenance of high dominance rank often important in the maintenance of territories and in involve frequent aggression moderated by conveying information among individuals in a rank testosterone which is considered as the quintessential order5. Scent marking frequency is correlated with 3 physiological moderator of such behaviour . Urine social dominance and the degree of aggressiveness in many mammalian species6. The scent marking ______communication system of cervids, which is poorly *Correspondence: E-mail: [email protected]; [email protected] understood at present, has been the subject of several Supplementary data available online at NOPR along with the article studies in recent years. The compounds produced by 782 INDIAN J EXP BIOL, NOVEMBER 2018

various integumentary structures, such as the biological factors of dominant/subordinate animals interdigital, preorbital, forehead, and tarsal glands are have been subjected to a few studies, but there is no either carried on the body or deposited onto the information on the chemistry of the signals and the environment on olfactory signposts7. In antelopes, both role of faecal testosterone. We, therefore, investigated males and females possess preorbital organs which the frequency of preorbital gland marking and its have a glandular region in a pouch adjacent to the nasal chemical composition in dominant males before, (medial) corner of the eye8,9. There is no clear evidence during and after the dominance hierarchy period and of a role across species for the preorbital gland10 but the compared the data with bachelor and subadult males. most reported role of preorbital secretions is in This study is the first step in understanding the territorial marking11. It has been already proposed that potential role of preorbital scent gland marking in the the preorbital gland (POG) marking behaviour is dominance hierarchy of Indian blackbuck. commonly associated with reproductive activities, and is performed more frequently by harem males and may Materials and Methods serve to advertise the presence of a territory and to Study area attract males11. The study was conducted in the Conservation and In blackbucks [Antelope cervicapra (L)], only adult Breeding Centre of Arignar Anna Zoological Park males establish territories that are consistently (AAZP) (13°16’S and 79°54’E at an altitude of MSL+ occupied year-around12. The males perform scent 10-100 m), Vandalur, Chennai, Tamil Nadu, India. marking from their well developed POG, while in The Park covers an area of about 510 hectares. The females these glands are vestigial or functionless2,13. habitat of AAZP is considered a tropical evergreen In addition, the territorial male gland marking can scrub, a degraded forest mostly consisting of thorny serve as permanent threat signals and help to control bushes. The average annual rainfall is about 250 mm the subordinate behavioural activities such as scent and the temperature is about 26ºC with little marking behaviour, scent production, and patrolling seasonal variation. activity, in order to maintain the hierarchy14. We have earlier studied the urine scent marking behaviour and Study animals its chemical constituents of male blackbucks and At the beginning of the study period, the blackbuck found that the frequency of urine marking is enclosure housed a total population of 75 animals. All significantly higher in dominant males with specific- blackbucks were housed in an outdoor enclosure of urinary volatiles during the hierarchy period as about 3.5 acres (1.4 hectares) within a dry moat. The compared to that of bachelor and sub-adult males15. group was classified, according to Rajagopal & 2 As many as 28 major urinary compounds identified in Archunan , into 14 adult males, 20 adult females, 16 which three unique volatile compounds were present subadult males, 18 subadult females and 6 immatures. only in the urine of dominant males during the The dominance hierarchy system was studied in the dominance-hierarchy period. These specific-volatiles 14 adult males only (4-7 year old). Each individual may play a major role in the defense of territories was recognized by the difference in the shape of the against potential intruders and to advertise their horn and other morphological features (Table 1). agonistic dominance over other males15. Water was offered ad libitum, whereas food was Histomorphology of POG in territorial and non- served twice a day (10.30 a.m. and 4.00 p.m.). These territorial male Indian blackbuck indicates are exactly the same blackbuck individuals and same considerable histological changes in sebaceous and observation periods used in previous study and apocrine secretory glands, which would produce obtained data on scent marking and chemical 15 pheromonal substance through POG for olfactory constituents of urine . communication9. The preorbital gland-marking behaviour and the Social status of male blackbuck chemical components of gland secretion have been The nomenclature of male social status are: (1) harem identified for a few antelope (Bovidae) species masters (i.e. dominant males) who hold harems; including Neotragus moschatus16; Damalicus dorca (2) challengers (i.e. subdominant males) without their phillipsi17; Raphicerus campestris18 and Ourebia own harems, but they challenge the harem master and ourebi19. Further, the physiological, social and try to hold females; and (3) bachelor groups which stay RAJAGOPAL et al.: ESTABLISHMENT OF DOMINANCE HIERARCHY IN INDIAN BLACKBUCK 783

Table 1 — Antelope cervicapra L. special identification characters of individual adult male blackbucks Animal no. & No. of twists Identification characters (age)# in horn 1 (7 yrs old) 4* Narrow horns with pointed tips; a large size mole in the lower abdomen on left side 2 (5 yrs old) 3* Almost parallel horns with pointed tips 3 (6 yrs old) 3* Left horn normal; right horn damaged and half bent 4 (7 yrs old) 4* Slightly twisted narrow horns with curved tips pointing each other 5 (5 yrs old) 3* Broad and compressed horn; tip of the right horn curved outward 6 (5 yrs old) 3* Long broad horns with curved tips outward 7 (6 yrs old) 3* Broad horns with their tips curved inward 8 (5 yrs old) 3* Long narrow horns with their tips curved inward 9 (5 yrs old) 3* Long broad horn; tip of the right horn damaged 10 (4 yrs old) 2* Narrow twisted horns, tip of the both horns very long and pointed 11 (5 yrs old) 3* Narrow horns, tip of the left horn little damaged 12 (5 yrs old) 3 ** Long twisted slim horn, tip of the right horn curved inward; right horn outward 13 (4 yrs old) 2* Long parallel horns with their tips straight onward sky 14 (4 yrs old) 2 * Broad horns; a large size scare in the right hind leg [* Thick solid horn; ** slim solid horn. #age given as per suggestion of zoo veterinarian] away from the female groups12. Indian blackbucks change their social status from ‘bachelor’ to ‘challenger’ and became ‘harem masters’; but some of them (bachelors) may never reach the highest social rank during their entire lifetime12.

Behavioural Observation

Aggressive behaviour In behavioral observations of the blackbuck, the aggressive acts between adult male blackbucks were recorded. Information about the aggressive act included the identity of the interacting adult male blackbucks, in order to know which adult male was the aggressor or dominant and which one was subservient or subordinate. Three types of aggression were recorded as follows: (i) Head up threats —movement of the head toward the opponent with neck and head held high, the horns thrown back, accompanied by a stiff-legged walk while the raised head is bobbed sideways. The ears are turned back and down, the tail is curled up and forward along the back exposing the rump patch; (ii) Chasing— this movement is usually instigated by a dominant male towards a subordinate male. If a subordinate male enters the territory of another during the estrus period, he will be chased; and (iii) Fighting —fights between two males Fig. 1 — Male blackbuck marking different substrates with i.e., dominant and pre-dominant trying to stake a claim. secretion from a preorbital gland. (A) marking a dry vertical stick; These fights are usually of a short duration; sometimes, (B) hanging parts; (C) bark of dead tree; (D) iron fence; (E) & (F) this has been in progress for a long time resulting in any horn scratching and deposition of secretion on park; (G) horn thrashing the food material; and (H) immediately deposits the one taking over the territory (i.e., leadership), but this gland secretion on food material. process need not be conclusive once for all. conspicuous, objects. The adult male blackbucks Scent marking behavioural observation carefully choose low, bare twigs of small bushes which The Indian blackbucks perform preorbital gland are systematically inserted into the open preorbital marking behaviour, aiming at specific, usually cavity by clear manoeuvring of the head (Fig. 1). 784 INDIAN J EXP BIOL, NOVEMBER 2018

The frequency of aggressive and preorbital gland Collection of preorbital gland secretion marking behaviours was observed in the 14 adult Samples of preorbital gland secretion were males adopting focal animal sampling method20. collected from dominant males before, during and The observations were made for 122 days after the formation of dominance hierarchy period and (18 months) from June 2007 to December 2008. also from bachelors and sub-adult males, for chemical The observation schedule was divided into two analysis. In the beginning, individual male samples shifts: morning 08.00-10.00 h and afternoon 14.00 to were analyzed separately for each category/status of 16.00 h, making it a total of 4 h of observation each male adopting GC-MS to find the chemical profiles, day. The animals were directly observed, using 8×40 and then samples from each male for each category binoculars, from the boundary of the enclosure. were pooled for GC-MS analysis. In the final analysis, the samples were pooled group-wise to Collection of faecal sample minimize the effect of individual variation [dominant Faecal samples were collected immediately after (n = 9), bachelor (n = 5) and sub-adult males (n = 5)]. observation of defecation of the identified adult males Preorbital gland secretion was collected from the and placing an amount equivalent to a handful into substrates (i.e., tree bark, sticks, grass, bushes, etc.) polythene bags using a wooden stick from 08.00 to and placed in a 2 mL vial. Once collected, the vials 10.00 h and 14.00 to 16.00 h. A sample was were labelled and placed in a cold thermo flask until considered to be fresh when the superficial layer of reaching the camp at 10.30 a.m. and 5.30 p.m., where faecal pellet was still wet and ensured that no insect the samples were placed in a freezer at 20ºC for contamination had occurred. Samples were placed in GC-MS analyses. The preorbital gland secretion was a thermo flask which occurred during 4 h after the collected taking assistance from the zoo veterinary sample collection. Faecal samples were transferred to surgeon and the standard guidelines of Central Zoo permanent freezing facilities and stored at 20°C for Authority, New Delhi, were followed. further analysis. GC-MS analyses Faecal extraction and hormone analysis Dichloromethane (DCM) was used as solvent in The faecal samples were lyophilized for 24 h GC-MS analysis. Portion (1 g) of the post materials (Model LGA05, MLW. Leipzig, Germany). Dried (i.e., Preorbital gland secretions) was homogenized faeces were then pulverized in a blender and all solid with PBS (pH 7.2) separately for each category. The inert materials (e.g., seeds and rough dietary fibre) homogenate was clarified by centrifugation at 12500 ×g were removed. Steroids were extracted according for 15 min and the supernatant was removed. Further, Archunan and Rajagopal21, with slight modification, the supernatant sample was taken from each animal in which a portion of the resulting powder (0.5 g) and it was mixed with 1 ml DCM (1:1 ratio) and was weighed and extracted with 5 mL of 80% filtered through a silica gel column (60-120 meshes) methanol. After vortexing for 30 s at high speed, the for 30 min at 37ºC. The filtered extract was cooled sample was shaken for 12 h on a mechanical shaker with liquid nitrogen to condense it to 1/5 of its and then centrifuged at 700 ×g for 15 min at 4C; the original volume. resulting supernatant was collected into a clean tube. The GC-MS analyses were made in QP-5000 An aliquot of the supernatant (100 µL) was diluted (Schimadzu, Japan). Two micro-litters of the extract 1:16 with assay buffer (20 mM Tris- was injected into GC-MS on a 30 m glass capillary hydroxyaminomethan, 0.3 M NaCl, 0.1% bovine column with a film thickness of 0.25 µm (30 m  0.2 mm serum albumin, and 0.1% Tween 80; pH 7.5) for i.d. coated with UCON 50 HB 2000) using initial radioimmunoassay (RIA) of faecal testosterone oven temperature of 40°C for 4 min, and increased using a commercial assay kit (Coat-A-Count Total to 250°C in steps of 15°C for 10 min. The gas Testosterone, DPC; Diagnostic Products chromatography apparatus (Schimadzu GC 15A) was Corporation, Los Angeles, CA, USA). All samples equipped with FID detector connected to an were assayed in duplicate in a single assay (the intra- integrator. The area under each peak was used for inter assay coefficient of variation was 8.4-13.1%, quantitative analysis. The detection accuracy was respectively). The sensitivity of testosterone assays 1 ng/peak. The relative amount of each component was 0.1%. was considered as the percent of the ion current. The RAJAGOPAL et al.: ESTABLISHMENT OF DOMINANCE HIERARCHY IN INDIAN BLACKBUCK 785

GC-MS was controlled by a computer at 70 eV using frequency of aggressive behaviour was studied in 14 ammonia as reagent gas at 95 eV for chemical adult males. The frequency range of aggressive ionization. Identification of unknown compounds was behaviour was recorded as 20.8±1.15 to 27.8±0.32 made by probability-based matching using the before the hierarchy period, 25.7±0.57 to 47.5±1.13 computer library built within the NICT 12 system. during the hierarchy period, and 8.1±0.31 to 29.0±0.31 after the hierarchy period in dominant Results males and 2.0±0.57 to 11.0±0.36 in bachelor males

Aggressive behaviour (Table 2). The frequency of aggressive behaviour Defensive and offensive aggressive behaviours was significantly higher (One-way ANOVA: F=0.001; occurred quite often for asserting dominance in male 3,59=56.34) in the dominant males during the blackbucks in the free ranging environment. The dominance period than before and after the

Table 2 — Antelope cervicapra L. Frequency (mean±SE) of aggressive behaviour of adult males (14 nos.) observed during the study period from June 2007 to December 2008 Dominancy period from June 2007 to December 2008

No. of 14 Jun. 18 July 31 Oct. 12-21 21 Feb. 03-22 23-27 28 Mar. 03-08 08-21 21-23 23 Apr. 04 May 29 Nov. 08-21 days -17 July -30 Oct. -12 Feb. Feb. -03 Mar. Mar Mar -02 Apr Apr Apr Apr -03 May -29 Nov -08 Dec Dec /animals (n=10) (n=20) (n=20) (n=5) (n=4) (n=7) (n=3) (n=4) (n=3) (n=7) (n=2) (n=6) (20) (n=5) (n=6) observed A.No: 1 5.9± 15.1± 19.6± BHP DHP* AHP BHP DHP* BAHP DHP* AHP 12.66± 8.7± 8.4± 8.2± 0.40 0.27 0.56 23.6± 31.6± 25.0± 25.66± 32.33± 27.33± 36.95± 13.33± 0.57 0.43 0.24 0.37 0.91$ 0.37$ 0.31$ 0.33$ 0.88$ 1.20$ 0.81$ 1.20$ A.No: 2 4.6± 9.8± 7.9± 5.2± 3.4± 6.6± 3.33± 2.0± 5.33± 4.8± 2.66± 5.0± 8.1± 3.8± 3.4± 0.47 0.38 0.27 0.37 0.24 0.40 0.33 0.57$ 0.33 0.37 0.66$ 0.57 0.23 0.58 0.40 A.No: 3 5.3± 6.0± 8.3± 5.4± 5.6± 5.4± 5.6± 3.0± 2.33± 3.8± 2.66± 5.33± 5.7± 3.6± 3.4± 0.51$ 0.25 0.21 0.24$ 0.24 0.24 0.33 0.57 0.33 0.37 0.66 0.33 0.26$ 0.40 0.40 A.No: 4 DHP* AHP 8.0± 5.4± 5.6± 2.2± 2.33± 2.0± 0.00± Died Died Died Died Died Died 37.2± 13.7± 0.27 0.24 0.24 0.20 0.33 0.57 0.00 0.59$ 0.33$ A.No: 5 BHP DHP* AHP 5.4± 5.4± 2.2± 2.33± 2.33± 2.33± 2.6± 2.33± 0.00± 0.00± Died Died 21.5± 42.33± 8.1± 0.24 0.24 0.20 0.33 0.33 0.33 0.40 0.33 0.00 0.00 0.51$ 0.43$ 0.31$ A.No: 6 7.8± 15.09± BHP DHP* BAHP DHP* AHP BHP DHP* BAHP DHP* AHP Died Died Died 0.46 0.49 27.8± 34.0± 29.0± 36.33± 20.66± 24.33± 29.5± 21.42± 25.7.0± Died$ 0.32$ 0.51$ 0.31$ 0.37$ 0.33$ 1.20$ 0.33$ 0.48$ 0.57$ A.No: 7 5.6± 8.2± 6.1± 3.6± 5.4± 3.6± 2.33± 3.5± 2.66± 3.6± 2.0± 2.6± 4.0± 3.8± 2.4± 0.40 0.20$ 0.23 0.40 0.24 0.40 0.33 0.5 0.33$ 0.40 0.00 0.40 0.36 0.37 0.24$ A.No: 8 10.9± BHP DHP* AHP 0.00± 0.00± 0.00± Died Died Died Died Died Died Died Died 0.58 22.2± 38.4± 0.00± 0.00 0.00 0.00 0.51$ 0.81$ 0.00$ A.No: 9 5.1± 8.3± 8.2± 3.2± 4.2± 3.0± 2.33± 3.0± 3.66± 6.3± 2.66± 3.4± 3.5± 2.4± 3.6± 0.37 0.30 0.20$ 0.37 0.58 0.37 0.66 0.57 0.33 0.46$ 0.88 0.24 0.26 0.24 0.40 A.No: 10 11.4± 12.9± 11.3± 8.4± 5.8± 8.0± 8.0± 8.33± 13.0± 15.4± 18.33± 12.0± 16.6± BHP DHP* 0.52 0.34 0.39 0.24 0.37 0.37 0.57 0.33 0.57 0.51 0.88 0.15 0.34 20.8± 35.25± 1.15$ 0.58$ A.No: 11 9.0± 11.1± 13.2± 18.6± 21.4± BHP DHP* AHP 18.0± 20.57± BHP DHP* AHP 13.4± 8.3± 0.33 0.34 0.55 0.40 0.51 25.6± 29.1± 15.0± 0.57 0.51 23.0± 35.8± 14.9± 0.54 0.40 0.40$ 1.45$ 1.0$ 1.15$ 1.52$ 0.58$ A.No: 12 10.4± 10.6± 12.25± 14.6± 13.4± 12.71± 8.66± 8.0± 8.0± 12.6± 15.0± 18.16± BHP DHP* AHP 0.42 0.49 0.55 0.51 0.51 0.37 0.66 0.57 0.57 0.24 0.15 0.87 21.5± 34.8± 12.8± 0.66$ 0.37$ 0.37$ A.No: 13 8.8± 8.1± 11± 6.2± 6.2± 5.6± 6.66± 3.0± 2.33± 5.4± 3.66± 3.66± 8.6± 5.6± 6.6± 0.35 0.34 0.36 0.58 0.58$ 0.40 0.66$ 0.57 0.33 0.24 0.66 0.66$ 0.22 0.40 0.40 A.No: 14 15.9± 12.8± 10.1± 10.4± 8.2± 10.6± 9.0± 12.0± 19.71± 13.8± 17.3± BHP DHP* AHP Died 0.45 0.35 0.25 0.24 0.37 0.40 0.57 0.57 0.60 0.58 0.51 25.83± 47.5± 0.00± 1.01$ 1.13$ 0.00$ [Animal numbers (A.No.) 2, 3, 7, 9 & 13 never reached the higher social rank during the study period, are considered as bachelor males. BHP: Before hierarchy period of dominant male; DHP: During hierarchy period of Dominant male; AHP: After hierarchy period of dominant male; BAHP: Both before or after hierarchy period of dominant male. Only values in $ were used for ANOVA. Values within parentheses (n) indicate the frequency of aggressive behaviour during the period of observation. * Statistically significant (P <0.05) as compared to bachelor males] 786 INDIAN J EXP BIOL, NOVEMBER 2018

dominance hierarchy period and in bachelor males (Tables S1 and S2. Suppl. data available online at NOPR along with the article separately). Moreover, the leadership of the adult male blackbuck was determined by fighting behaviour, which is one of the important aggressive behaviours (Fig. 2). It is remarkable to note that during determination of dominance, the fighting occurred between the dominant and pre-dominant individuals, which caused injury to both and sometimes leading to death (Fig. 3).

Duration of dominance Among the 14 adult male blackbucks observed under the semi-natural captive conditions, 9 were found to have established dominance hierarchies during the study period from June 2007 to December 2008 (Table 3). In the beginning the animal number 4 (14th June to 17th July 2007) exhibited dominance and superseded the other males - animal numbers 5, 8, 6, 1, 11, 14, 12 and 10. Animal number 14 remained dominant in the same enclosure for the longest duration - 210 days, from 4th May 2008 to 29th November 2008. Subsequently, animal numbers 6, 1, and 11 became dominant (animal number 6 four times; Fig. 2 — Attacking behaviour exhibited by male during dominance animal number 1 three times and animal number 11 two determination. (A) & (B) a clash initiates the pushing; (C) & (D) face times); however, the duration of dominance of these each other with head low during a pause; (E) & (F) necks and head animals ranged for only 3 to 20 days. Animal numbers turn as horn remain locked at bases; (G) and (H) a male turns horns 2, 3, 7, 9, and 13 never reached the higher social pushing to opponent, the opponent neck locked his antler. ranking (dominant or pre-dominant) during the entire study period. In the present study, the number of females mated by dominant males during the specified observation periods was also recorded. The animal number 14 mated with more females than the other dominant males (Table 3). However, animal numbers 10 and 12 did not mate with any female during their hierarchy period.

Preorbital gland scent marking behaviour The frequency of preorbital gland marking behaviour was observed in 14 adult males. The preorbital gland marking frequencies were recorded as follows: 8.5±0.86 to 13.85±0.33 before the hierarchy formation period; 12.0±1.38 to 17.65±0.73 during the hierarchy formation period; 2.4±0.75 to 11.28±0.83 after the hierarchy formation period in the dominant males and 2.33±0.21 to 5.5±0.45 in bachelor males (Table 4). One-way ANOVA with post hoc DMRT comparison test Fig. 3 — During dominant determination fighting occurred resulting showed that the average preorbital gland scent marking in (A) animal number 6 died due to severe injury near diaphram, frequency was significantly higher (F=49.79; df=3,59; (B) in animal number 8 leg was injured; and (C) in animal number 14 P <0.001) during the formation of hierarchy period eye was severely injured and finally the animal died. RAJAGOPAL et al.: ESTABLISHMENT OF DOMINANCE HIERARCHY IN INDIAN BLACKBUCK 787

Table 3 — Antelope cervicapra L. Duration of dominancy of the 9 dominant males from June 2007 to December 2008 Animal Duration of dominancy Days Mated Remarks number From To (No.) females (No.) 4 14 June 2007 17 July 2007 35 1 Death due to injury in internal organ (12 April 2008) 5 18 July 2007 30 Oct. 2007 105 8 Death due to injury in internal organ (3 December 2008) 8 31 Oct. 2007 12 Feb. 2008 104 6 Death after leg injury (30 March 2008) 6 12 Feb. 2008 21 Feb. 2008 10 1 1 21 Feb. 2008 3 Mar. 2008 11 0 6 03 Mar. 2008 22 Mar. 2008 20 3 11 23 Mar. 2008 27 Mar. 2008 5 0 1 28 Mar. 2008 2 Apr. 2008 6 1 6 03 Apr. 2008 8 Apr. 2008 6 1 1 08 Apr. 2008 21 Apr. 2008 14 2 6 21 Apr. 2008 23 Apr. 2008 3* 1 Death due to injury during infighting (23 April 2008) 11 23 Apr. 2008 3 May 2008 12 1 14 04 May 2008 29 Nov. 2008 210** 11 Death due to eye injury during infighting (17 December 2008) 12 29 Nov. 2008 8th Dec. 2008 11 0 10 08 Dec. 2008 21st Dec. 2008 14 0 [*Minimum duration of dominance; ** Maximum duration of dominance]

Table 4 — Antelope cervicapra L. Frequency (mean±SE) of preorbital gland marking behaviour of adult males (n=14) observed during the study period (days) from June 2007 to December 2008

Dominancy period from June 2007 to December 2008 No. of 14 Jun. 18 July 31 Oct. 12-21 21 Feb. 03-22 23-27 28 Mar. 03-08 08-21 21-23 23 Apr. 04 May 29 Nov. 08-21 days -17 July -30 Oct. -12 Feb. Feb. -03 Mar. Mar Mar -02 Apr Apr Apr Apr -03 May -29 Nov -08 Dec Dec /animals observed (n=10) (n=20) (n=20) (n=5) (n=4) (n=7) (n=3) (n=4) (n=3) (n=7) (n=2) (n=6) (20) (n=5) (n=6) A.No: 1 4.9± 6.25± 7.15± BHP DHP* AHP BHP DOMI* BAHP DHP* AHP 5.83± 4.6± 5.4± 5.0± 0.50 0.51 0.31 10.2± 12.75± 8.42± 9.33± 13.25± 10.0± 14.28± 4.0± 0.54 0.27 0.98 0.89 0.80$ 0.47$ 0.52$ 0.66$ 1.37$ 1.15$ 0.92$ 1.00$ A.No: 2 5.5± 4.61± 4.1± 4.2± 5.0± 3.85± 3.66± 4.0± 2.66± 4.14± 3.0± 4.0± 3.8± 2.6± 2.33± 0.45 0.36 0.29 0.49 0.81 0.59 0.66 0.57$ 0.33 0.59 0.00$ 0.68 0.26 0.24 0.21 A.No: 3 4.8± 5.1±0.4 4.85± 5.0± 3.0± 3.85± 3.0± 2.5± 2.66± 3.14± 4.0±1.0 4.33± 3.15± 2.4± 3.66± 0.44$ 3 0.31 0.00$ 0.00 0.40 0.00 0.28 0.33 0.34 0 0.66 0.25$ 0.24 0.42 A.No: 4 DHP* AHP 6.75± 4.6± 4.5± 3.57± 2.66± 3.0± 1.66± Died Died Died Died Died Died 14.9± 6.15± 0.31 0.75 0.95 0.36 0.33 0.00 0.33 1.16$ 0.46$ A.No: 5 BHP DHP* AHP 4.6± 4.0± 3.0± 3.0± 4.0± 3.66± 3.0± 3.0± 4.33± 1.85± Died Died 12.0± 17.65± 4.8± 0.75 0.57 0.00 1.00 0.57 0.66 0.37 0.00 0.42 0.15 1.07$ 0.73 0.32$ A.No: 6 7.8± 8.5± BHP DHP* BAHP DHP* AHP BHP DHP* BAHP DHP* AHP Died Died Died 0.75 0.48 12.05± 13.4± 8.5± 14.15± 6.66± 9.25± 12.33± 11.28± 12.5± Died$ 0.37$ 0.92$ 0.86$ 0.85$ 0.66$ 1.10$ 1.45$ 0.83$ 2.50$ A.No: 7 5.3± 5.0± 4.75± 3.8± 4.5± 4.71± 2.66± 3.25± 3.0± 5.0± 2.5± 4.33± 3.2± 3.0± 3.33± 0.57 0.34$ 0.37 0.49 0.95 0.52$ 0.33 0.62 0.00$ 0.43 0.50 0.42 0.25 0.00 0.33 A.No: 8 9.1± BHP DOMI* AHP 2.5± 2.14± 2.0± Died Died Died Died Died Died Died Died 0.67 13.85± 16.25± 2.4± 0.5 0.50 0.00 0.33$ 0.44$ 0.75$ A.No: 9 4.6± 4.75± 4.7± 4.2± 3.0± 4.42± 3.66± 4.0± 2.66± 3.85± 4.0± 3.33± 4.3± 5.0± 3.66± 0.47 0.25 0.30$ 0.80 0.00 0.36 0.66 0.57 0.33 0.59$ 1.00 0.55 0.33 0.89 0.42 A.No: 10 6.3± 6.35± 6.0± 3.8± 3.5± 5.28± 4.33± 4.0± 3.66± 5.0± 2.0± 5.66± 6.05± 11.00± DHP* 0.65 0.37 0.22 0.80 0.51 0.52 0.66 0.57 0.66 0.43 0.00 0.42 0.21 1.18$ 13.33± 0.84$ A.No: 11 6.5± 7.05± 7.35± 3.8± 5.75± BHP DHP* 4.75± 5.33± 5.71± BHP DHP* AHP 5.0± 4.66± 0.73 0.41 0.43 0.49 1.10 10.28± 12.66± 0.25$ 0.66 0.18 11.5± 13.5± 3.3± 0.89 0.61 0.77$ 0.66$ 3.51$ 1.06$ 0.27$ (contd.) 788 INDIAN J EXP BIOL, NOVEMBER 2018

Table 4 — Antelope cervicapra L. Frequency (mean±SE) of preorbital gland marking behaviour of adult males (n=14) observed during the study period (days) from June 2007 to December 2008 (contd.)

Dominancy period from June 2007 to December 2008 No. of days 14 Jun. 18 July 31 Oct. 12-21 21 Feb. 03-22 23-27 28 Mar. 03-08 08-21 21-23 23 Apr. 04 May 29 Nov. 08-21 -17 July -30 Oct. -12 Feb. Feb. -03 Mar. Mar Mar -02 Apr Apr Apr Apr -03 May -29 Nov -08 Dec Dec /animals observed (n=10) (n=20) (n=20) (n=5) (n=4) (n=7) (n=3) (n=4) (n=3) (n=7) (n=2) (n=6) (20) (n=5) (n=6) A.No: 12 4.4± 5.0± 5.9± 4.6± 4.0± 5.85± 5.66± 5.0± 3.0± 4.71± 6.0± 8.0± BHP DHP* AHP 0.42 0.39 0.20 0.98 1.00 0.40 0.66 0.81 0.00 0.52 1.00 0.44 11.7± 12.0± 6.33± 0.49$ 1.38$ 0.61$ A.No: 13 3.9± 4.1± 4.0± 3.4± 3.5± 3.75± 3.66± 4.0± 2.66± 2.42± 4±1.00 4.33± 4.6±0.3 3.0± 4.66± 0.54 0.22 0.22 0.40 0.5$ 0.36 0.66$ 0.57 0.33 0.20 0.66$ 7 0.54$ 0.61 A.No: 14 6.3± 6.35± 7.6± 6.2± 5.75± 5.28± 5.66± 4.0± 5.66± 7.57± 10.0± BHP DHP* AHP Died 0.44 0.31 0.27 0.73 1.10 0.52 0.66 0.57 0.66 0.36 0.00 13.16± 16.2± 0.00± 1.14$ 0.73$ 0.00$ [Animal numbers (A.No.) 2, 3, 7, 9 & 13 never reached the higher social rank during the study period, are considered as bachelor males. BHP: Before hierarchy period of dominant male; DHP: During hierarchy period of Dominant male; AHP: After hierarchy period of dominant male; BAHP: Both before or after hierarchy period of dominant male. Only values in $ were used for ANOVA.Values within parentheses (n) indicate the frequency of preorbital gland scent marking during the period of observation. * Statistically significant (P <0.05) as compared to bachelor males] (13.96) followed by before (10.27) and after (5.11) the before, during and after the formation of dominance formation of hierarchy period in the dominant males as hierarchy (Figs. 4 A-C), bachelor males (Fig. 4D) and compared to bachelor males (3.94) (Tables S3 and S4). sub-adult male (Fig. 4E). The peak numbers refer to the list of compounds showed in Table 6. Tentative Faecal testosterone levels identification of the volatile organic compounds in the The faecal testosterone level varied considerably secretion was based on a comparison with data in among the 14 adult males. The range of faecal mass spectra libraries. Forty three major compounds testosterone levels in dominant males during the were, thus, identified in preorbital gland secretion of hierarchy period was between 90.0±8.67 to dominant males before, during and after the hierarchy 143.5±21.56 ng/g; before hierarchy period 48.5±4.19 formation periods and in bachelor and subadult males. to 91.0±4.01 ng/g; and after hierarchy period The identified compounds belonged to carboxylic 27.5±2.57 to 59.5±5.82 ng/g and bachelor males acid (16 volatiles), alcohol (11 volatiles), alkane 29.0±1.00 to 56.0±2.23 ng/g (Table 5). The one-way (8 volatiles), alkene (4 volatiles), aldehyde ANOVA with post hoc comparison (DMRT) test (2 volatiles) and ketone (2 volatiles) groups. Among clearly showed that the average faecal testosterone the 43 volatile compounds, 39 were identified in the concentration was significantly higher (F=34.68; dominant male during the hierarchy formation period, df=3,59; P <0.001) in the during the formation of 34 before the hierarchy formation period, 27 after the hierarchy period (100.18 ng/g) followed by before hierarchy formation period; 25 compounds were (74.68 ng/g) and after (45.87 ng/g) the formation of identified in bachelor males and 13 in subadult males. hierarchy period in the dominant males as compared Interestingly, 9 detectable compounds, namely to bachelor males (32.81 ng/g) (Tables S5 and S6). undecanoic acid, (E)-5-tetradecane, dodecanoic acid, The dominance hierarchy among the 14 adult 1-chloro-octadecane, nonadecanoic acid, 1-1-oxybis males in semi-natural condition was perfectly linear. decane, 1-docosene, tetracosane, and octacosane, The frequency of preorbital gland scent marking were found in the preorbital gland secretion of behaviour (smb), number of mating attempt (nma) dominant male before, during and after the hierarchy and duration of dominance (smb: r = 0.97; nma: formation period. These compounds were absent in r=0.95, P <0.001 and ft: r=) were directly related to bachelor and sub-adult males. Sequentially, four dominant males (Fig. S1. Available online at NOPR specific compounds namely, 2-methyl propanoic acid along with the article separately). (I), 2-methyl-4-heptanone (II), 2,7-dimethyl-1-octanol (III) and 1,15-pentadecanediol (IV) were found in the Chemical profiles of preorbital gland secretion preorbital gland secretion of only the dominant male The profiles of volatile compounds in the preorbital during the hierarchy formation period. The gland secretion of dominant male were obtained compounds, cis-1,13-cyclopentanediol, nonanoic acid, RAJAGOPAL et al.: ESTABLISHMENT OF DOMINANCE HIERARCHY IN INDIAN BLACKBUCK 789

Table 5 — Antelope cervicapra L. Level of faecal testosterone (ng/g dry weight: mean±SE) in adult males (n=14) observed during the study period from June 2007 to December 2008 Dominancy period from June 2007 to December 2008

No. of 14 Jun. 18 July 31 Oct. 12-21 21 Feb. 03-22 23-27 28 Mar. 03-08 08-21 21-23 23 Apr. 04 May 29 Nov. 08-21 days -17 July -30 Oct. -12 Feb. Feb. -03 Mar. Mar Mar -02 Apr Apr Apr Apr -03 May -29 Nov -08 Dec Dec /samples 3 times 5 times 4 times 2 times 2 times 2 times 1 time 1 time 1 time 2 times 1 time 2 times 5 times 2 times 2 times collected per animal A.No: 1 23.33± 31.6± 56.75± BHP DHP* AHP BHP DHP* BAHP DHP* AHP 59.5± 44.6± 39.5± 46.5± 4.41 3.11 1.10 76± 95.5± 57.2± 82$ 92.5$ 83.7$ 104.5± 61.5$ 5.82 1.73 1.50 1.50 11.03$ 17.5$ 9.52$ 21.56$ A.No: 2 23± 26± 36.5± 34.5± 27± 23± 25 26.5$ 28 37± 34$ 36.5± 49± 41.5± 46.5± 4.58 2.23 1.55 1.50 2.00 2.00 2.00 1.50 2.90 1.50 8.25 A.No: 3 35± 34.4± 25.75± 23.5± 29± 22.5± 30 32 32.5 34± 34 36.5± 35.5± 40.5± 32.5± 6.03 3.51 2.17 1.50$ 1.00 0.50 1.00 1.50 0.81$ 5.51 2.50 A.No: 4 DHP* AHP 35.5± 31.5± 33.5± 21± 25 20.52 20 Died Died Died Died Died Died 90± 34.8± 3.52 3.51 1.50 1.00 8.67$ 2.96$ A.No: 5 BHP DHP* AHP 36± 29± 29.5± 30 25 25.7 22± 22.75 20.5± 18.8± Died Died 78± 137.2± 45.25± 1.00 1.00 1.50 2.00 1.50 1.50 10.61$ 8.30$ 3.77$ A.No: 6 50.33± 63.4± BHP DHP* BAHP DHP* AHP BHP DHP* BAHP DHP* AHP Died Died Died 9.53 2.60 68.5± 80.5± 84.5± 99.2± 53$ 78.36$ 90$ 85.5± 87.15$ died 4.19$ 15.54$ 5.51$ 22.5$ 4.01$ A.No: 7 37± 30.2± 36.25± 35.5± 37± 22.5± 25 25 26.5$ 29± 31 41.5± 32.6± 33± 37.5± 2.64 0.86$ 2.32 0.50 4.01 2.50 1.00 0.50 1.46 2.00 4.75$ A.No: 8 66± BHP DHP* AHP 22.5± 25.5± 20 Died Died Died Died Died Died Died Died 3.79 75.2± 112.75± 28.5± 2.50 2.57 3.28$ 14.36$ 1.50$ A.No: 9 41.33± 31.2± 35.0± 37± 32.5± 33.5± 32 32.5 33.12 34± 36 31.5± 40.4± 36.5± 34± 3.28 1.16 2.04$ 1.00 2.50 2.50 1.00$ 1.50 2.29 1.50 4.01 A.No: 10 36.66± 43.4± 43.25± 37.5± 37.5± 41± 37 38 40 48± 52.5 56.5± 63± BHP DHP* 6.64 1.97 1.37 2.50 2.50 6.00 3.00 1.50 2.72 87± 97± 9.20$ 5.01$ A.No: 11 48.66± 58.4± 56± 57.5± 64± BHP DHP* AHP 72 82± BHP DHP* AHP 50± 42± 9.41 2.66 1.58 1.50 4.01 79.5± 85$ 36.5$ 10.02 85$ 103.5± 48.6± 5.01 1.00 9.52$ 11.53$ 3.98$ A.No: 12 39.66± 45± 44.5± 47.5± 42.5± 33± 45 43.7 45 48± 51.6 66.5± BHP DHP* AHP 4.18 1.84 2.02 2.50 0.50 2.00 2.00 1.50 82.8± 95.5± 52.5± 7.65$ 21.56$ 7.52$ A.No: 13 36.33± 39.8± 34.25± 39± 34± 41± 38 35 42 42.5± 38 40± 42.5± 40± 39± 2.96 1.65 2.17 1.00 4.01$ 1.00 2.50 5.01$ 2.50 5.01 1.00 A.No: 14 40.66± 44± 46.0± 42.5± 48.5± 46.5± 48 48 51 56.5± 62.5 BHP DHP* AHP Died 3.18 0.63 1.08 2.50 4.42 1.50 1.50 74.5± 133.5± 0.00± 12.20$ 7.39$ 0.00$ [Animal numbers (A.No.) 2, 3, 7, 9 & 13 never reached the higher social rank during the study period, are considered as bachelor males. BHP: Before hierarchy period of dominant male; DHP: During hierarchy period of Dominant male; AHP: After hierarchy period of dominant male; BAHP: Both before or after hierarchy period of dominant male. Only values in $ were used for ANOVA. Values within parentheses (n) indicate the number of samples collected during the period of observation. *Statistically significant (P <0.05) as compared to bachelor males]

(Z)-9-tetradecenoic acid, 1,14-tetradecanediol, different stages, dominant, bachelor and sub-adult hexadecanoic acid and tricosane appeared before males. By contrast, a few compounds, viz., 6-methyl- and during the hierarchy formation period 1-heptanol, 1,9-nonanediol, 2-butyl-1-octanol, but disappeared after the hierarchy period. 1-heptadecanol, pentacosane and heptacosane were Tridecanoic acid, pentadecanoic acid, 1-octadecanol, found both in dominant male (all three different eicosene, (Z)-9-octadecenoic acid and acetic acid stages) as well as bachelor males but absent in the octadecyl ester were identified during the all the three sub-adult males. 790 INDIAN J EXP BIOL, NOVEMBER 2018

territories22. In this study, we used aggressive behaviour as reflection of dominance hierarchy in the blackbuck herd, during the hierarchy formation period when the dominant males had the highest score of aggressive behaviours such as threatening, chasing and fighting. This aggressive behaviour was the one which controlled the feeding, patrolling and scent marking activities of the opponents. In this respect, the high frequency of aggressive behaviour could confer an advantage in competition for determining hierarchy and it is reported in the different mammals such as red deer23, white-tailed deer24 and musk deer25. Data from the dominant male red deer26 show that aggression and reproductive efforts decline after subordinate stage, while another study shows increased effort (aggressive interaction/tending frequency) from the subordinate and then again reach dominant stage27. Hence, we suggest that the dominant male blackbuck occupies areas rich in food, and unleashes aggressive attack on subordinates, preventing them from feeding so as to deprive them from feeding an extra quantity. So that they would tend to grow more, and thereby gain strength to attain and maintain dominance position. Coming to the mating performance of the remaining males animal number 14 mated with the largest number of females compared to the other dominant males. It was also observed that during the breeding season there was stiff competition among the males to take to dominance and, therefore, the duration of dominance was comparatively short. It was found that the high ranking males mated with more females per year than lower ranking males. Simmons and Holley28 found a great frequency of mating and offspring production by higher ranking males. Reappearance (i.e., repeated occurrence) of Fig. 4 — GC-MS profiles of volatile compounds in the preorbital gland secretion of dominant male (A) before; (B) during; (C) after dominance and the formation of hierarchy were the formation of dominance hierarchy period; (D) bachelor; and observed in animal numbers 6, 1 and 11 from (E) sub-adult male blackbucks. Peak numbers are those referred in February to April through for a short period (A. no. 6: Table 6. 3-20 days; A. no. 1: 6-14 days; A. no. 11: 5-12 days). All the above compounds identified in the It is reported that the ownership (dominance) change- preorbital gland secretion of dominant, bachelor and over is common during mating period and after a sub-adult males had molecular weight between 88 and period the erstwhile dominant males attempts to 2 394. The GC analysis showed that the compounds fall regain its previously held territory . Such attempts between the retention times of 5-45 min. most often lead to serious fights, and injuries are 2,12 common . In our study, though during the rut Discussion periods (Aug-Oct and Mar-May) a male lost hierarchy Agonistic behaviours have been used in many (i.e., rank), and it was re-established subsequently. In types of social interactions particularly to establish addition, more females exhibited heat during these either dominance-subordinate hierarchies or exclusive periods (Table 3). It is found that sudden change in RAJAGOPAL et al.: ESTABLISHMENT OF DOMINANCE HIERARCHY IN INDIAN BLACKBUCK 791

Table 6 — Antelope cervicapra L. Volatile compounds tentatively identified in the preorbital gland secretion of dominant and subordinate male Blackbucks Peak No. Compound name Nature of compound Dominant male Subordinate male BHP DHP AHP BM SAM 1 2-methyl propanoic acid Carboxylic acid - + - - - 2 2-methyl-4-heptanone Ketone - + - - - 3 Cis-1,3-cyclopentanediol Alcohol + + - + - 4 4-heptanol Alcohol + + - + + 5 6-methyl-1-heptanol Alcohol + + + + - 6 2,7-dimethyl-1-octanol Alcohol - + - - - 7 Decanal Aldehyde + + - + + 8 Nonanoic acid Carboxylic acid + + - - - 9 1,9-nonanediol Alcohol + + + + - 10 (Z)-6-dodecen-1-ol Alcohol + + + + - 11 Decanoic acid Carboxylic acid - - - - + 12 Undecanoic acid Carboxylic acid + + + - - 13 2-butyl-1-octanol Alcohol + + + + - 14 (E)-2-tetradece-1-ol Alcohol + + + - - 15 Dodecanoic acid Carboxylic acid + + + - - 16 Tetradecanal Aldehyde - - - + - 17 Tridecanoic acid Carboxylic acid + + + + + 18 (Z)-9-tetradecenoic acid Carboxylic acid + + - + - 19 1,14-tetradecanediol Alcohol + + - - - 20 Tetradecanoic acid Carboxylic acid - + + + - 21 Pentadecanoic acid Carboxylic acid + + + + + 22 1-hexadecanol Alcohol + + + + + 23 1,15-pentadecanediol Alcohol - + - - - 24 Hexadecanoic acid Carboxylic acid + + - - - 25 1-hepadecanol Alcohol + + + + - 26 Heptadecanoic acid Carboxylic acid - - - + - 27 1-octadecanol Alcohol + + + + + 28 Eicosane Alkane + + + + + 29 (Z)-9-octadecenoic acid Carboxylic acid + + + + + 30 Octadecanoic acid Carboxylic acid + + + + + 31 Cyclononadecanone Ketone + + + + + 32 1-chlorodecane Alkane + + + - - 33 10-undecenoic acid Carboxylic acid + + + + + 34 Nonadecanoic acid Carboxylic acid + + + - - 35 1,1-oxybis-decane Alkane + + + - - 36 Docosene Alkene - - - + - 37 1-docosene Alkene + + + 38 Acetic acid, octadecyl ester Carboxylic acid + + + + + 39 Tricosane Alkane + + - - - 40 Tetracosane Alkane + + + - - 41 Pentacosane Alkane + + + + - 42 Heptacosane Alkane + + + + - 43 Octacosane Alkane + + + - - Total 34 39 27 25 13 [Abbreviation: + present, - absent, BHP - before hierarchy period, DHP – during hierarchy period, AHP – after hierarchy period, BM – bachelor male, SAM – sub-adult male]

792 INDIAN J EXP BIOL, NOVEMBER 2018

dominance is associated with the decreased male chimpanzees. It is argued that testosterone may aggressiveness, hormone levels, physical strength, be associated with the frequency or intensity of male natural horn shedding or change of breeding season29. aggression and/or with dominance rank in the Therefore, the present results clearly indicate that the presence of receptive females during mating seasons formation and maintenance of dominance hierarchy is as well as in situation of social instability38. Our essential for establishing superiority in mating and results lead to the conclusion that testosterone leaving behind offspring. strongly associates with formation of hierarchy and, The frequency of preorbital gland marking more importantly with changes in the subordinate behaviour by the dominant male during formation of stage. Further, the correlation among testosterone hierarchy period as observed in the present study is level, preorbital gland marking frequency, mating significant. This is consistent with the observation in activity and duration of leadership period are other ungulates like Alces alces gigas30, Dama dama31 interlinked in the dominance. and Oreotragus oreotragus32. It is to be noted that Among the 43 preorbital gland secretory volatiles 34 similar high frequency of urinary marking behaviour were present before, 39 during and 27 after the exhibited by dominant male blackbuck during the dominance hierarchy period in the dominant males; 25 hierarchy period is recorded in our previous study15. were present in bachelor and 13 in sub-adult males. In primates, the dominant female’s scent-marking Qualitatively also the volatiles differed between odours inhibit the reproductive cyclycity in dominant and subordinate males. Since all the subordinate females33. The higher frequency of scent subordinate males in this study were 1.5 to 2 year old marking behaviour is directly associated with high and all dominant males were 4 to 7 year old, we levels of gonadal steroids and it facilitates attraction speculate that the difference in stage-related nature of of the opposite sex and advertises the territorial the compounds is associated with the age or dominance ownership and competitive ability34,35. In the present status. Available reports indicate that many of the study the dominant males often preferred particular volatile compounds are hormone-dependent4,39. The places close to the food trough and peripheral region preorbital gland, in producing pheromonal substances, of territory for their preorbital gland marking (Fig. S2. would play a role in the determination and/or Available online at NOPR along with the article establishment of social hierarchy. Thus, the appearance separately). It indicates that the dominant male of four specific-volatile compounds [i.e., 2-methyl generally uses the preorbital gland secretion as propanoic acid (I), 2-methyl-4-heptanon (II), 2,7- reliable signal for territory demarcation and to warn dimethyl-1-octanol (III) and 1,15-pentadecanediol other males from entry to the territorial region. (IV)]observed from the dominant males’ preorbital Therefore, the present results suggest that the gland secretion during his hierarchy period may preorbital gland scent marks (including behaviour and represent behaviourally important chemical signals and volatile compounds) play an important role in the suggest that these compounds may have role in female protecting the territories by preventing the intruders attraction and/or male-male aggression. and to show their dominance authority. This investigation is a first to report in reference to The level of testosterone regulates many aspects of identification of volatile compounds in the preorbital male reproductive physiology36. For example, gland of male blackbuck. Significant variations in testosterone secretion is correlated with many aggressive behaviour, preorbital gland scent marking, behavioural traits particularly mating competition faecal testosterone and chemical profiles of preorbital including reproductive motivation, territoriality, mate gland secretion are found in the dominant males before, guarding, heightened aggression and display during and after the hierarchy period when compared behaviour37. In the present study, high concentration with bachelor and sub-adult males. The results in regard of faecal testosterone was noted in the dominant to behavioural observation and chemical profiles males during the formation of hierarchy period obtained from the present and previous15 studies, provide followed by before and after the formation of circumstantial evidence that the blackbuck may use both hierarchy period compared to bachelors. Similar preorbital gland secretion and urine for territorial observation was observed by Muehlenbein and marking, to attract members of the opposite sex for Watts36 when the fecal testosterone levels were mating and to repel members of the same sex which directly associated with dominance rank in wild adult would conspire to reach higher social hierarchy. The RAJAGOPAL et al.: ESTABLISHMENT OF DOMINANCE HIERARCHY IN INDIAN BLACKBUCK 793

present study convincingly concludes that the specific 11 Gosling LM, Scent marking in an antelope lek territory. volatile compounds from the preorbital gland, exhibition Anim Behav, 35 (1987) 620. 12 Ranjitsingh MK, The Indian Blackbuck (Natraj publisher, of behaviours and higher level of testosterone appears to Dehradun), 1989, 1. be important during the formation of dominancy in male 13 Rajagopal T, Rajkumar R, Ponmanickam P, Achiraman S, blackbuck. Padmanabhan P & Archunan G, Identification of pheromone- carrying protein in the preorbital gland post in the Acknowledgement endangered Indian male Blackbuck Antelope cervicapra L. Indian J Exp Biol, 53 (2015) 771. We thank Chief the Wildlife Warden and the 14 Rajagopal T, Manimozhi A, Archunan G, Diurnal variation Director of the Arignar Anna Zoological Park (AAZP), of preorbital gland marking behaviour of captive male Indian Vandalur, Chennai, for granting permission to carry out Blackbuck (Antelope cervicapra L.) and its territorial this study. The authors also thank Dr K Senthilkumar, significance. Biol Rhy Res, 42 (2011) 27. Dr PN Khan and Dr. R Thirumurugan of the AAZP 15 Rajagopal T, Archunan G, Geraldine P & Balasundaram C, Assessment of dominance hierarchy through urine scent for help in collection of preorbital gland secretion. TR marking and its chemical constituents in male blackbuck acknowledges the UGC for the award of RGNF and Antelope cervicapra, a critically endangered species. Behav SERB - Young Scientist, New Delhi; GA Proc, 85 (2010) 58. acknowledges the UGC, New Delhi for the award of 16 Stander MA, Burger BV & Le Roux M, Mammalian UGC-BSR Faculty Fellowship. The facilities availed exocrine secretions. XVII: Chemical characterization of preorbital secretion of male suni, Neotragus moschatus. from UGC-SAP Phase II and DST-FIST is gratefully J Chem Ecol, 28 (2002) 89. acknowledged. 17 Burger BV, Nell AE, Spies HSC, Le Roux M & Bigalke RC, Mammalian exocrine secretions. XII. Constituents of References preorbital gland secretions of bontebok, Damaliscus dorcas 1 Koyama NF, Ronkainen K & Aureli F, Durability and dorcas and blesbok, D. d. phillipis. J Chem Ecol, 25 (1999a) flexibility of chimpanzee grooming patterns during a period 2085. of dominance instability. Am J Primatol, (2017) e22713. 18 Burger BV, Greyling J & Spies HSC, Mammalian exocrine https://doi.org/10.1002/ajp.22713. secretions. XIV: constituents of preorbital secretion of 2 Rajagopal T & Archunan G, Chemical communication in steenbok, Raphicerus campestris. J Chem Ecol, 25 (1999b) Indian Blackbuck (Antelope Cervicapra L) with special 2099. reference to dominance. In: Chemical Signals in Vertebrates 19 Mo WP, Burger BV, Le Roux M & Spies HSC, Mammalian 13, (Eds. Schulte BA & Goodwin T; Springer-Verlag exocrine secretions: IX. Constituents of preorbital gland International, Switzerland), 2016, 217. secretion of oribi, Ourebia ouribi. J Chem Ecol, 21 (1995) 3 Nelson AC, Cunningham CB, Ruff JS & Potts WK, Protein 1191. pheromone expression levels predict and respond to the 20 Altmann J, Observational study of behaviour: sampling formation of social dominance networks. J Evol Biol, 28 methods. Behaviour, 22 (1974) 377. (2015) 1213. 21 Archunan G & Rajagopal T, Detection of estrus in Indian 4 Novotny MV, Harvey S & Jemiolo B, Chemistry of male blackbuck: Behavioural, hormonal and urinary volatiles dominance in the house mouse, Mus domesticus. Experientia, evaluation. Gen Comp Endocrinol, 181 (2013) 156. 16 (1990) 109. 22 Ibáñez A, Menke M, Quezada G, Jiménez-Uzcátegui G, 5 Massen JJ, Slipogor V & Gallup AC, An observational Schulz S & Steinfartz S, Diversity of compounds in femoral investigation of behavioral contagion in common marmosets secretions of Galápagos iguanas (genera: Amblyrhynchus (Callithrix jacchus): Indications for contagious scent- and Conolophus), and their potential role in sexual marking. Front Psychol, 6 (2017) 1190. communication in lek-mating marine iguanas 6 Vaglio S, Minicozzi P, Romoli R, Boscaro F, Pieraccini G, (Amblyrhynchus cristatus) Peer J, 5 (2017) 1. Moneti G & Moggi-Cecchi J, Sternal gland scent-marking 23 Albon SD, Staines HJ, Guinness FE & Clutton-Brock TH, signals sex, age, rank, and group identity in captive Density-dependent changes in the spacing behavior of female mandrills. Chem Senses, 41 (2016) 177. kin in red deer. J Anim Ecol, 61 (1992) 131. 7 Gassett JW, Wiesler DP, Baker AG, Osborn DA, Miller KV, 24 Townsend TW & Bailey ED, Effects of age, sex and weight Marchinton LR & Novotny M, Volatile compounds from the on social rank in penned white-tailed deer. Am Midland Nat, forehead region of male white-tailed deer (Odocoileus 106 (1981) 92. virginianus). J Chem Ecol, 23 (1997) 569. 25 Meng X, Cody N, Gong B & Xiang L, Stable fighting 8 Muller-Using D & Schloeth R, Das Verhalten der Hirsche. strategies to maintain social ranks in captive male Alpine Kukenthal. Handb Zool, 10 (1967) 1. musk deer (Moschus sifanicus). Anim Sci J, 83 (2012) 617. 9 Rajagopal T & Archunan G, Histomorphology of preorbital 26 Yoccoz NG, Mysterud A, langvatn R & Stenseth NC, Age gland in territorial and non-territorial male blackbuck and density- dependent reproductive effort in male red deer. Antelope cervicapra, a critically endangered species. Proc R Soc Lond Ser B, 269 (2002) 1523. Biologia, 66 (2011) 370. 27 Komers PE, Pelabon C & Stenstron D, Age at first 10 Gray DR, Flood PF & Rowell JE, The structure and function reproduction in male fallow deer: age-specific versus of muskox preorbital glands. Can J Zool, 67 (1989) 1134. dominance-specific behaviours. Behav Ecol, 4 (1997) 456. 794 INDIAN J EXP BIOL, NOVEMBER 2018

28 Simmons LW & Holley R, Offspring viability benefits but no males in a free-living root vole (Microtus oeconomus) apparent costs of mating with high quality males. Biol Lett, 7 population. Physiol Behav, 128 (2014) 26. (2011) 419. 38 Surbeck M, Deschner T, Schubert G, Weltring A & 29 French JA, Mustoe1 AC, Cavanaugh J & Birnie AK, The Hohmann G, Mate competition, testosterone and intersexual influence of androgenic steroid hormones on female relationships in bonobos, Pan paniscus. Anim Behav, 83 aggression in ‘atypical’ mammals. Philos Trans R Soc Lond (2012) 659. B Biol Sci, 368 (2013) 1. 39 Miller KV, Marchinton RL, Forand KJ & Johanesen KL, 30 Bowyer RT, Van Ballenberghe V & Rock KR, Scent Dominance, testosterone level, and scraping activity in a marking by Alaskan moose: characteristics and spatial captive herd of white-tailed deer. J Mammal, 68 (1987) 812. distribution of rubbed trees. Can J Zool, 72 (1994) 2186. 40 Andersson G, Brundin A & Andersson K, Volatile compounds 31 Massei G & Bowyer RT, Scent marking in fallow deer: from the interdigital gland of reindeer (Rangifer t. tarandus L.). effects of lekking behaviour on rubbing and wallowing. J Chem Ecol, 5 (1979) 321. J Mammal, 80 (1999) 633. 41 Wood WF, Volatile components in metatarsal glands of sika 32 Roberts SC & Dunbar RIM, Female territoriality and the deer, Cervus Nippon. J Chem Ecol, 29 (2003) 2729. function of scent marking in a monogamous antelope 42 Riddle HS, Riddle SW, Rasmussen LEL & Goodwin TE, (Oreotragus oreotragus). Behav Ecol Sociobiol, 47 (2000) 417. First disclosure and preliminary investigation of a liquid 33 Smith TE & Abbott DH, Behavioural discrimination between released from the ears of African elephants. Zoo Biol, 19 circumgenital odor from peri-ovulatory dominant and (2000) 475. anovulatory female common marmosets (Callithrix jacchus). Am J Primatol, 46 (1998) 265. 43 Raymer J, Wiesler D, Novotny MV, Asa C, Seal US & 34 Novotny MV, Jemiolo B, Wiesler D, Ma,W, Harvey S, Xu F, Mech LD, Chemical scent constituents in urine of wolf Xie TM & Carmack M. A unique urinary constituent, (Canis lupus) and their dependence on reproductive 6-hydroxy-6-methyl-3-heptanone, is a pheromone that hormones. J Chem Ecol, 12 (1986) 297. accelerates puberty in female mice. Chem Biol, 6 (1999) 377. 44 Giblin-Davis RM, Gries R, Gries G, Pen-Rojas E, Pinzon I, 35 Ferkin MH, The response of rodents to scent marks: Four Pana JE, Perez AL, Pierce HD & Oehlschlager AC, broad hypotheses. Horm Behav, 68 (2015) 43. Aggregation pheromone of palm weevil, Dynamis borassi. 36 Muehlenbein MP & Watts DP, The costs of dominance: J Chem Ecol, 23 (1997) 2287. testosterone, cortisol and intestinal parasites in wild male 45 Ross GN, Fales HM, Lloyd HA, Jones T, Sokoloski EA, chimpanzees. Biopsychosoc Med, 4 (2010) 2. Marshall-Batty E & Blum MS, Novel chemistry of 37 Borowski Z, Malinowska A & Ksiazek A, Relationships abdominal defensive glands of Nymphalid butterfly, Agrulis between dominance, testosterone level and scent marking of vanilla. J Chem Ecol, 27 (2001) 1219.