Indian Journal of Experimental Biology Vol. 39, September 200 I, pp. 887-891

Rat cheek gland compounds: Behavioural response to identified compounds

S Kannan* & G Archunan Department of Animal Science. School of Life Sciences, Bharathidasan University. Tiruchirappalli 620 024, India

Received /0 November 2000; revised 9 April 2001

Cheek g land secretions from sexually mature and reproductively active male and female laboratory rats (Wister strain). Rattlls non•egicus albinus were analysed by gas chromatognphy/mass spectroscopy. Alkanes, aliphatic acids esters and alcohols were identified in the secretions. Cheek gland preparation from males contained predominantly three compounds, viz. di-n-octyl phthalate (I); I ,2- benzene diacarboxylic acid butyl (2 -ethyl hexyl) ester (II ): and 1.2 benzene dicarboxylic acid (2-methylpropyl) ester (Ill) whereas in the cheek gland of females two compounds, viz, 1,2-benzene dicarboxylic acid (2-methylpropyl) ester (I); and 2,6.1 0 dedecatrien-1-o I, 3,7, I 1-trimethyi-(Z, E) (II) were the major fractions. The identified compounds were tested for odour preferences. Compounds II and III of males and I and II of females attracted both male and female conspecifics. By contrast, n mpound I from males only attracted females. The level of attraction also varied from compound to compound. The results suggest that cheek gland secretions have pheromonal functions.

Chemical signals are involved in olfactory communication in many animals. Among mammals, olfactory communication has been especially well studied, primarily focusing on scent gland secretions 1

-5,

urinary pheromones6·7

, faeces8-10 and vaginal

secretions 11• Such odourous substances can send

powerful messages that modulate social and reproductive behaviours of mammals. The study of perceptual mechanisms of pheromonal cueing has been hampered by the lack of identification of specific compounds functioning as conspecific chemical cues. Only for a few species of mammals such as mice, hamsters and elephants the behavioural, chemical and neurobiological aspects of pheromonal communication related to sexual reproduction have been investigated 10

'12

. Laboratory rat has a variety of known and potential sources of scents such as integumentary glands including preputial, cheek, armpit and flank glands and excretory substances namely urine, faeces and vaginal secretion 13

•14

• A variety of functions for secretions of skin glands have been described , such as territorial marking, maintenance of social and reproductive behaviours, individual recognition and evocation of fighting behaviour and regulation of ovulation in humans 15

•17

•

Scent glands are comparatively larger and more active in males than in females. In several mammalian

*Present address: Department of Zoology & Microbiology, Ayya Nadar .lanaki Ammal College (Autonomous), Sivakasi 626 124, India E.mail: skanmbt2000@yahoo.com

species, the scent gland development and the acttvtttes are androgen-dependent5

&18

• Volatile compounds are predominant in scent gland secretions; non-volatile compounds are less evident. These volatile and non-volatile compounds are easily detected by the main or accessory olfactory systems of the responding animals 19

-21

. The cheek gland secretions are deposited on substrates including branches of trees, or food items, or on the body itself. Chemical identification of mammalian urinary pheromones have been achieved in mice22

-24

, tiger25

and elephane 2. Though sebaceous glands play an

important role in the maintenance of social and reproductive behaviours in mammals, especially in rodents, adequate information is not available about the chemistry of their secretions. In the present study, an attempt to determine the chemical nature of cheek gland secretions was determined . The behavioural importance of the identified compounds was ascertained in an effort to delineate their functions .

Materials and Methods

Sexually mature and reproductively active adult male and female laboratory rat (Wister strain), Rattus norvegicus albinus weighing 200 to 250 g were purchased from the King's Institute, Chennai and housed individually 111 polypropylene cages (40x25xl5 em) with 2.0cm husk as bedding material. The bedding material was changed twice a week. Rat feed (Hindustan Lever Ltd, Bangalore) and water was provided ad libitum. Animals were maintained on a l4L: 100 photoperiodic schedule in

888 fNDIAN J EXP BIOL, SEPTEMBER 2001

a climate controlled environment with a temperature of 25° ± 3°C. Lights were on from 0600 to I800 hrs.

Twenty-five females in estrus and 25 male rats were sacrificed by cervical dislocation under mild di­ethyl ether anesthesia. At autopsy, cheek glands were dissected out under a dissection microscope and placed in double distilled solvent mixture (n-hexane and dichlormethane 1 : I ratio v/v) and ground well separately for about I 0 min with glass homogeniser under ice cold condition . The supernatant was filtered through silica gel (50-60!1 mesh size) and collected in a glass vial and sealed with air tight screw type cap made up of glass (Borosil). The sample vials were stored at -20°C until they were used for analysis.

GC-MS analysis was made in a Shimadzu QP5000 (Japan) instrument under computer control at 70 eV. Using ammonia as reagent gas at 95-eV (Ref. 24) performed chemical ionization . The identified compounds were fractionated by the method of Pause era/ 26

.

The extracted samples (20mL) were di tilled for 5-10 min at room temp.erature (30"C) under a vacuum of 0.2 torr. The distillate was condensed by cooling with liquid nitrogen. These distillates were restored to their original volume (2mL) vaccum condensation . Y -maze odour preference test was conducted by following the procedure of Ferkin and Seamon27

.

Three individuals were (randomly taken from a pool of 20 rats) used for each behaviour analysis. Fresh samples were used for each trial. The behaviour was assessed for 15 min with the identified compounds

and the solvent mixture was used as control. Both male and females were tested. The time taken for visiting each fraction was recorded. The data were analyzed by student's t test28

.

Results The gas chromatographic study revealed the

presence of eleven different compounds in male cheek gland extract. Three compounds constituted the major part of the extract. In the male cheek gland, the identified compounds were di-n-octyl pthalate (I); I ,2-benzene dicarboxylic acid butyl (2-ethyl hexyl) ester (II); and 1,2 benzene dicarboxylic acid (2-rnethylpropyl) ester (III). In the female cheek gland, only two compounds were observed, namely 1,2-benzene dicarboxylic acid (2-methyl propyl) ester (I); and 2,6, I 0 dodecatrien-1-o 1 ,3,7, 11-trimethyl- (Z,E) (II). Compound I of the female was also identified in the male cheek gland (Figs I, 2)

The odour preference test revealed that male cheek gland extract attracted both male and female conspecifics. Compound I of male was more attractive to females than to males whereas, compound II and III attracted both male and female rats (Table I). Compound I and II of female cheek gland have strongly attracted both males and females and female responders spent significant amount of time with compound I and II of the female cheek gland. Among the two major fractions of female cheek gland, compound II was significantly more attractive to both males and females than compound I.

Table 1-Behavioural respon ses of male and female laboratory rats to compounds (I-III) identified from cheek gland extract ( 15 min odour preference tests)

[Values are mean± SE of 6 observations)

Responders

Same sex Opposite sex

Same sex Opposite sex

ss

109.66±2.14# 406.14±2.68*

216.43±2.38# 210.86±3.50#

BS

26.00± 1.56 28.00± 1.82

28.31 ± 1.65 27.43± 1.05

II ss BS

Male cheek gland extract

224.33 ± 1.84# 29.71 ± 1.63 226.7 I ± 2.35# 32.40±2.34

Female cheek gland extract

371.57±3.'72# 29.00± 1.33 364.66 ± 4.57# 26.30± 1.61

SS - Scented slide (Solvent+ Solute); and BS- Blank slide/ control slide (Solvent only) * Opposite sex attractant; and# both sex altractant

Ill ss BS

204.71 ±4.80# 30.60±3.00 213.16±3.1 1# 24.57 ± 1.37

Male cheek gland extract: Compound I (Di-n-octyl phthalate); compound II (I ,2-benzene dicarboxylic acid butyl(2-ethylhexyl) ester ); and compound III ( 1.2-benzene dicarboxylic acid (2-methylpropyl) ester). Female cheek gland extract: Compound I (1,2-benzene diacarboxylic acid (2-methylpropyl) ester ); and compound II (2.6, 10 dodecatrien-1-ol ,3.7, 11-trymethyl-(Z.E)).

KANNAN & ARCHUNAN: RAT CHEEK GLAND COMPOUNDS 889

Discussion The present study has revealed that male and

female cheek gland extracts consist of eleven major compounds (see GC and Mass spectra Figs 1 ,2). Earlier chemical studies in mammalian species, such as mouse29

·30

, hamster31, tiger 25 and elephant 32

showed that pheromones consist of group of compounds, and the major compound are of greater biological significance. Dodecyl propionate is identified in the preputial glands of female rat pups and is involved in attracting other members33

. The

>­f-Vi z UJ t­z

UJ

> ~ _J

UJ a::

dorsal gland of Tayassu tajacu (collared red peccary) contains some volatile fractions which are involved in the maintenance of social behaviour34

.

The results of the behavioural tests with identified compounds in the cheek gland are given in Table 1. The behavioural importance of cheek gland secretions are well documented in bandicoot rats24

, black tailed deer35

, arboreal squirrels36 and shrews37. These studies

suggest that the secretary activity of cheek glands and the frequency of scent marking are higher in adult males than in females and the secretions elicit social

2 3

20 22 24. 26 28 30 32 34 36 38 4.0

100

UJ

LJ 41 69 ~ z UJ LJ a: UJ a.. UJ

> ~ ....)

UJ 0::

6 r l9s 121 149 oL j II

100

56 76

65 I so

76

50

149 207

200

189

RETENTION TIME

279 Oi-n-oc tyl phthalate 410 400

305 231249 332349 372 390

300 4.00 500

2, 6, 10 -Dodecatrien -1- ol, 3,7,11- tr imethyl-, !Z, f l 402

523 541

233

200

284 I

I 6~

367 I 452 5~3 564 Jill 64~ 300 400 500 600 700

93

1, 2-Benzen 104

121

100 150 200

1. 2- Benzened icarboxylic acid, bis !2- methylpropyl l ester 104

205 222

121 135 149 160

250

100 150 200 250

MASS/CHARGE RATIO (m/e)

Fig. !-Capillary column (with a film thickness of 0.25 m) chromatogram of the rat cheek gland extract. Each compound-giving rise to a signal is analyzed by mass spectrometry (Coupled GC-MS, Shimadzu, Japan). In mass spectra, ion relative abundance is plotted against ion ratio of charge to mass ( m/e). All mass spectra were analyzed and the chemical structures determined by comparison with computerized databases.

890 INDIAN 1 EXP BIOL, SEPTEMBER 2001

Di- n-octylphthalate

1. 2 Benzene dicarboxylic acid bis (2-methyl propyl )ester

~OH

2,6,10 -Dodecotrien-1-ol, 3.7. 11-trimethyi!Z. E)

~ OXOl fQT -o~

1,2-Benzene dicarboxylic acid butyl-2-ethylhexyl ester

Fig. 2-Chemical structure of the compounds idemified in rat cheek gland extracts.

interactions in both the sexes37. The present findings

demonstrate that cheek gland secretions are involved in the attraction of both male and female rat 14

• The response of male to female scent was comparatively greater than that of the female to male cheek gland secretions. This may be due to the active involvement of androgen in pheromonal communication in rats5

.

Kannan 14 recently reported that male rat preputial gland contains a mixture of volatiles including di-n­octyl phthalate. Di-n-octyl phthalate is also identified in the male cheek gland of laboratory rats. Further, the female responders devote more time investigating this com ound. Hence, thi s compound may also be involved in sexual attraction between males and females. Earlier reports23 are consistent wi th th is view. Since di-n-octyl phthalate is found only in male

cheek glands and is a powerful attractant for females it may be considered as an important pheromonal compound in the rat.

Acknowledgement We thank Professor. C.J. Dominic, formerly of the

Banaras Hindu University, and the Management of Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi for encouragement, Prof. L.E.L. Rasmussen, Oregon Graduate Institute, USA for critical reading of the manuscript and SPIC Science Foundation, Chennai for analytical work. We gratefully acknowledge the CSIR, New Delhi for financial assistance.

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