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Occurrence of flehmen in male buffaloes (Bubalus bubalis) with special reference to estrus
Theriogenology 61 (2004) 861–866
Occurrence of flehmen in male buffaloes (Bubalus bubalis) with special reference to estrus Swamynathan Rajanarayanan, Govindaraju Archunan* Department of Animal Science, Bharathidasan University, Tiruchirappalli 620024, India Received 14 April 2003; received in revised form 3 July 2003; accepted 4 July 2003
Abstract Pheromonal cues present in the urine and vaginal mucus during the estrus period elicit courtship behaviour in mammals including the characteristic flehmen behaviour. In the present study, the flehmen behaviour was assessed in male buffaloes by exposing them to heifers for three consecutive estrous cycles. Behavioural observations revealed that the bulls sniffed the female’s external genitalia, responded to the chemical signal(s) and exhibited the flehmen behaviour similar to those reported in other ungulates. The flehmen behaviour was recorded daily during 15-min contact with females for three consecutive estrous cycles. Out of 180 observations during five days estrus periods (3 to þ1 days) which included high frequency of flehmen behaviour and out of 637 observations with or without flehmen behaviour during diestrus periods, the bull displayed 365 and 441 flehmen behaviour, respectively. Average numbers of all (2:03 0:66) and repeated flehmen (1:05 0:64) behaviour during estrus periods were significantly higher than those of diestrus periods of all (0:69 0:25) and repeated flehmen (0:11 0:10). The statistical significance was higher (P < 0:001) in repeated flehmen behaviour of estrus as compared to that in diestrus. In case of all flehmen behaviour, the statistical significance was higher (P < 0:01) in estrus when compared to that in diestrus. The occurrence of flehmen behaviour in male buffaloes clearly indicates that the specific pheromonal compound(s) present in the urine/vaginal mucus during estrus significantly influence the flehmen behaviour. # 2003 Elsevier Inc. All rights reserved. Keywords: Estrous cycle; Pheromonal cues; Urine; Flehmen; Buffalo
1. Introduction Chemical cues (pheromones) reportedly play a major role in mammalian reproduction and behaviour [1,2] and are found to be significant in the induction of ovulation, as well as, * Corresponding author. Tel.: þ91-431-2407040; fax: þ91-431-2407045. E-mail address: [email protected] (G. Archunan).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.07.004
862
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866
in identifying the receptivity of the female. The sources of chemical signals are urine, faeces, vaginal secretions and specialized scent glands [2,3]. Communication of the timing of the physiological event of ovulation and coordination of sexual behaviour are important for ensuring successful fertilization [4,5]. While the existence of sexual pheromones and their significance in reproductive behaviour are well known in laboratory mammals, there is little information about them in domestic and farm animals. Chemical cues have been studied in some detail in cows. No information is, however, available on this aspect of reproduction for buffaloes. The female reproductive system is generally complicated due to variation in the endocrine response, and this is more so in buffalo reproduction. The main problem in buffalo reproduction is the occurrence of silent ovulations without any behavioural signs of estrus [6], thereby, making the detection of heat in buffaloes more difficult than in cattle [7]. It is well known that the signs of estrus mainly manifest the receptivity of females during the estrous cycle. The problem of estrus detection in cows is overcome by using the herd bulls which monitor olfactory and gustatory cues routinely [8,9]. The visual and acoustic stimuli of the cow are important for optimal detection of estrus by the bull [10,11]. Bulls receive the estrus specific chemical signal from females and then exhibit flehmen behaviour. This is preceded by fast tongue strokes over the rostra and medial part of the palate which have separate innervations and yield strokes that actually trigger the flehmen response [12]. During flehmen, the attractants from the female enter the olfactory organ of the bull which, in turn, relays the signals to the hypothalamic areas of the brain [13]. The ‘flehmen behaviour show’ is the most definite and integral part of the premating scenario. Bioassays to quantify the pheromone activity have been designed and defined using the flehmen behaviour of bull [14]. In the present study, we have postulated that the flehmen behaviour of the buffalo bull in response to estrus (3 to þ1 day) and diestrus stages of female, by virtue of its ability to elicit the presence of estrus-indicating pheromones, could be used as a practical tool for detection of estrus. To our knowledge, a study of this nature has not been carried out in buffaloes so far.
2. Materials and methods The behavioural study was carried out in the Government Livestock Farm, Orathanadu, Tanjore District, South India. Twelve Bulls and twelve heifers (Bubalus bubalis) were used in the study. The animals were fed a standard diet and water was allowed ad libitum. Rectal examination of each heifer was performed regularly at one or two weeks intervals to verify the normal morphological changes in the internal organs. The flehmen behaviour was studied on the bulls under natural conditions of breeding during three consecutive estrous cycles (from 12 to þ12 days with day 0 estrus) including 36 estrous cycles in all. A total of 817 observations (180 estrus days and 637 diestrus days) was made during the experiment. The duration of each observation was approximately 15 min. During the observation period, the bull was allowed to investigate the female and the frequency of flehmen behaviour of each bull to each female was recorded throughout the estrous cycle. Generally, the bull sniffed the genital regions of the female and raised the neck, extended
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866
863
Fig. 1. Male displaying the flehmen behaviour after inhaling the pheromonal substance(s) from the vaginal region of female.
the chin and inhaled with slightly opened mouth, the tongue being held in a flat position and the upper lip curled so that the nostrils became partly closed (Fig. 1). The bull also manipulated the tongue during the flehmen behaviour. The statistical analysis was done by applying Student’s ‘t’ test on the data concerning the flehmen behaviour in estrus (3 to þ1) and diestrus [15].
3. Results The exhibition of flehmen behaviour by the buffalo bulls observed in the present study appears to be typical for buffalo reproduction. The flehmen behaviour was observed throughout the 36 estrous cycles as presented in Fig. 2. When the bull was released to investigate the female, he first inhaled the pheromonal substance(s) from the vaginal region by sniffing and elicited flehmen behaviour. When the bull identified the estrus specific compound(s) during the first flehmen behaviour, he continued smelling and displaying flehmen frequently known as ‘repeated flehmen behaviour’. If he did not find the specific compound(s) during investigation, he stopped with a single flehmen or without flehmen. In all, 817 observations consisting of 180 estrus (3 to þ1) and 637 diestrus days were made. The flehmen behaviour was exhibited in 806 comprising 365 in estrus and 441 in diestrus days. A total of 258 repeated flehmen behaviour was observed including a high frequency of 188 flehmen (72.9%) during estrus and 70 (27.1%) during diestrus. Average numbers of all (2:03 0:66) and repeated flehmen (1:05 0:64) behaviour during estrus periods were
864
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866 3.5
3
ALL FLEHMEN Flehmen numbers
2.5
REPEATED FLEHMEN
2
SIGNIFICANT ALL AND REPEATED FLEHMEN DURING ESTRUS PERIODS p<0.01 & p<0.001 RESPECT IVELY WHEN COMPARED WITH DIESTRUS
1.5
1
0.5
0
-12-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0
1 2
3 4 5 6 7
8 9 10 11 12
Days
Fig. 2. Occurrence of flehmen during estrous cycle (day ‘0’ estrus).
significantly higher than those of diestrus periods of all (0:69 0:25) and repeated flehmen (0:11 0:10). The statistical significance was higher (P < 0:001) in repeated flehmen behaviour of estrus as compared to that in diestrus. In case of all flehmen behaviour, the statistical significance was higher (P < 0:01) in estrus when compared to that in diestrus.
4. Discussion In mammals other than primates, males proceed to mate after receiving the specific signals from females. The females generally produce such signals from early estrus. The use of biological assay for simultaneous detection of estrus related odors seems to be a prerequisite for the chemical study of pheromones [14]. Flehmen is a unique behaviour shown by bulls and it could be used as a parameter for monitoring the estrous cycle [14]. It has been suggested that the flehmen behaviour takes place under the stimulation of the vomeronasal organ through which male ungulates discriminate estrus and non-estrus urine [16]. Interestingly, during the observation periods, the flehmen behaviour increased from day –4, peaked at day 0 (3:11 1:19) and decreased in late estrus. The present findings in buffaloes support the earlier reports in cows in that the flehmen behaviour is most frequent on the day of estrus and decreases during the diestrus phase [14]. Our observations on the flehmen behaviour of the buffalo bull to the females the day before estrus and throughout the estrus are similar to the report made on cows [17]. The present investigation clearly shows that estrus elicited significant repeated flehmen behaviour. Obviously, the occurrence of flehmen during estrus is due to the presence of specific pheromonal cue(s) present in the female urine/vaginal mucus. It is also interesting to note that even though diestrus elicited repeated flehmen behaviour (0:11 0:10, significantly lower P < 0:001 when compared to estrus), it may also be controlled by
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866
865
certain sex pheromones. The presence of such pheromones, however, may be negligible and may have connection with some other events of estrous cycle, such as, interovulatory follicle growth [14]. The flehmen behaviour during the diestrus stages, nevertheless, had either singled or was non-responsive. It is well known that when bulls identify the specific pheromonal compound(s) they will continue to exhibit repeated flehmen and whenever such specific compound(s) are not identifiable, the bulls will stop with single flehmen [14,16]. Pheromonal compound(s) present in estrus provoke the manifestation of premating behaviour in the males. The males have the capability to discriminate between estrus and diestrus urine based on chemical cues present in it [18]. The flehmen is triggered with a high frequency through inhaling the estrus urine and sniffing the vaginal mucus. The sex pheromone believed to be present in the excretory products is released into the environment and perceived by the bull which eventually evokes the behavioural response. This is consistent with other studies reported in cows [14,19,20], sheep [21,22], cotton- top tamarins [5], equines [23] and elephants [24]. It is well known that the pheromonal compounds, particularly those involved in reproductive behaviour are mediated through the vomeronasal organ (VNO) which is situated in the nasal septum [25,26]. Therefore, it is also our future endeavor to find out the functional aspect of VNO in perception of chemical signal in relation to expression of flehmen in male buffaloes. The general concept of females to express their readiness for mating by producing specific olfactory signals is supported by the results of this study in the case of buffaloes. Since estrus specific chemical signal(s) is primarily involved in inducing the flehmen behaviour it can be concluded that specific pheromonal compound(s) present in urine/vaginal mucus are responsible for the flehmen behaviour and this can be used as a simple technique for detection of estrus in buffaloes. Further characterization of urinary/vaginal mucus pheromonal compound(s) in buffaloes is underway in our laboratory.
Acknowledgements We thank Dr. C. Natarajan formerly of the Indian Veterinary Research Institute, Izatnagar for his constant encouragement and critical reading of the manuscript. This investigation was supported by grants from the Department of Science and Technology, Government of India.
References [1] Keverne EB. Pheromones and sexual behaviour. In: Money J, Musaph H, editors. Handbook of sexology. North Holland: Elsevier Biomedical Press; 1977. p. 413–28. [2] Dominic CJ. Chemical communication in animals. J Sci Res (Banarus Hindu University) 1991;41:157–9. [3] Aron C. Mechanisms of control of the reproductive function by olfactory stimuli in female mammals. Physiol Behav 1979;59:229–84. [4] Schams D, Schallenberger E, Hoffman B, Karg H. The oestrus cycle of the cow: hormonal parameters and time relationships concerning oestrus, ovulation and electrical resistance of the vaginal mucus. Acta Endocrinol 1977;86:180–92.
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[5] Ziegler ET, Epple G, Snowdon CT, Porter TA, Belsher MA, Kuderlink KI. Detection of the chemical signals of ovulation in the cotton-top tamarin, Saguinus oedipus. Anim Behav 1993;45:313–22. [6] Dobson H, Kamonpatana M. A review of female cattle reproduction with special reference to comparison between buffaloes, cows and Zebu. J Reprod Fertil 1983;77:1–36. [7] Danell B, Gopakumar N, Nair MCS, Rajagopalan K. Heat symptoms and detection in surti buffalo heifers. IJAR 1984;5:1–7. [8] Williamson NB, Morris RS, Blood DC, Cannon CM, wright PJ. A study of oestrous behaviour and oestrous detection methods in a large commercial dairy herd. 2. Oestrous signs and behaviour patterns. Vet Rec 1972;91:58–62. [9] Reinhardt V. Flehmen, mounting, copulation among members of a semi wild cattle herd. Anim Behav 1983;31:641–50. [10] Blaschke CF, Thompson Jr DL, Humes PE, Godke RA. Olfaction, sight and auditory perception of mature bulls in detection estrual responses in beef heifers. 10th Int Congr Anim Reprod AI 1984;3:284–6. [11] De Vyst A, Thines G, Henriet L, Soffie M. Influence des stimulation auditives sur le comportement sexual du taureau. Experientia 1964;20:648–50. [12] Jacobs VL, Sis RF, Coppock CE. Tongue manipulation of the palate assists estrous detection in the bovine. Theriogenology 1980;13:353–6. [13] Vandenbergh JG. Pheromones in mammals. In: Encyclopedia of reproduction. London, UK: Academic Press; 1999. p. 764–9. [14] Hradecky P, Sis RF, Klemm WR. Distribution of flehmen reactions of the bull throughout the bovine estrous cycle. Theriogenology 1983;20:197–204. [15] Zar JH. Biostatistical analysis. 2nd ed (Englewood Cliffs, NY: Prentice-Hall; 1984. p. 360. [16] Estes RD. The role of vomeronasal organ in mammalian reproduction. Mammalia 1972;36:315–42. [17] Blazquez NB, French JM, Long SE, Perry GC. Bovine oestrous odours: behavioural and histological investigations. 11th Int Congr Anim Reprod AI 1988;4:550–2. [18] Hart GH, Mead SW, Regan WM. Stimulating the sex drive of bovine males in artifical insemination. Endocrinology 1946;39:221–3. [19] Dehnhard M, Claus R, Pfeiffer S, Schopper D. Variation estrus related odors in the cow and its dependency on the ovary. Theriogenology 1991;35:645–52. [20] Rameshkumar K. Chemical characterization of bovine (Bos taurus) urine with special reference to reproductive behaviour. Ph.D. Thesis. Bharathidasan University, Tiruchirappalli, Tamil Nadu, India; 2000. [21] Blissitt MJ, Bland KP, Cottrell DF. Olfactory and vomeronasal chemoreception and the discrimination of oestrous and non-estrous ewe urine odours by the ram. Appl Anim Behav Sci 1990;27:325–35. [22] Blissitt MJ, Bland KP, Cottrell DF. Detection of estrous-related odour in ewe urine by rams. J Reprod Fertl 1994;101:189–91. [23] Ma W, Klemm WR. Variations of equine urinary volatile compounds during the oestrous cycle. Vet Res Comm 1997;21:37–446. [24] Rasmussen LEL, Lee TD, Zhang A, Roelofs WL, Dews Jr GD. Purification, identification concentration and bioactivity of (2)-7-dodecen-1-yl acetate: sex pheromone of the female Asian elephant, Elephas maximus. Chem Senses 1997;22:417–38. [25] Dominic CJ. Primer pheromonal influence on reproduction in rodents. Indian Rev Life Sci 1987;7:21–49. [26] Wysocki CJ. Vomeronasal chemoreception: its role in reproductive fitness and physiology. In Neural control of reproductive function. In: Lakoski JM, Perez-Polo JR, Rassin DK, editors. New York: Alan R. Liss; 1984. p. 545–66.
Occurrence of flehmen in male buffaloes (Bubalus bubalis) with special reference to estrus Swamynathan Rajanarayanan, Govindaraju Archunan* Department of Animal Science, Bharathidasan University, Tiruchirappalli 620024, India Received 14 April 2003; received in revised form 3 July 2003; accepted 4 July 2003
Abstract Pheromonal cues present in the urine and vaginal mucus during the estrus period elicit courtship behaviour in mammals including the characteristic flehmen behaviour. In the present study, the flehmen behaviour was assessed in male buffaloes by exposing them to heifers for three consecutive estrous cycles. Behavioural observations revealed that the bulls sniffed the female’s external genitalia, responded to the chemical signal(s) and exhibited the flehmen behaviour similar to those reported in other ungulates. The flehmen behaviour was recorded daily during 15-min contact with females for three consecutive estrous cycles. Out of 180 observations during five days estrus periods (3 to þ1 days) which included high frequency of flehmen behaviour and out of 637 observations with or without flehmen behaviour during diestrus periods, the bull displayed 365 and 441 flehmen behaviour, respectively. Average numbers of all (2:03 0:66) and repeated flehmen (1:05 0:64) behaviour during estrus periods were significantly higher than those of diestrus periods of all (0:69 0:25) and repeated flehmen (0:11 0:10). The statistical significance was higher (P < 0:001) in repeated flehmen behaviour of estrus as compared to that in diestrus. In case of all flehmen behaviour, the statistical significance was higher (P < 0:01) in estrus when compared to that in diestrus. The occurrence of flehmen behaviour in male buffaloes clearly indicates that the specific pheromonal compound(s) present in the urine/vaginal mucus during estrus significantly influence the flehmen behaviour. # 2003 Elsevier Inc. All rights reserved. Keywords: Estrous cycle; Pheromonal cues; Urine; Flehmen; Buffalo
1. Introduction Chemical cues (pheromones) reportedly play a major role in mammalian reproduction and behaviour [1,2] and are found to be significant in the induction of ovulation, as well as, * Corresponding author. Tel.: þ91-431-2407040; fax: þ91-431-2407045. E-mail address: [email protected] (G. Archunan).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.07.004
862
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866
in identifying the receptivity of the female. The sources of chemical signals are urine, faeces, vaginal secretions and specialized scent glands [2,3]. Communication of the timing of the physiological event of ovulation and coordination of sexual behaviour are important for ensuring successful fertilization [4,5]. While the existence of sexual pheromones and their significance in reproductive behaviour are well known in laboratory mammals, there is little information about them in domestic and farm animals. Chemical cues have been studied in some detail in cows. No information is, however, available on this aspect of reproduction for buffaloes. The female reproductive system is generally complicated due to variation in the endocrine response, and this is more so in buffalo reproduction. The main problem in buffalo reproduction is the occurrence of silent ovulations without any behavioural signs of estrus [6], thereby, making the detection of heat in buffaloes more difficult than in cattle [7]. It is well known that the signs of estrus mainly manifest the receptivity of females during the estrous cycle. The problem of estrus detection in cows is overcome by using the herd bulls which monitor olfactory and gustatory cues routinely [8,9]. The visual and acoustic stimuli of the cow are important for optimal detection of estrus by the bull [10,11]. Bulls receive the estrus specific chemical signal from females and then exhibit flehmen behaviour. This is preceded by fast tongue strokes over the rostra and medial part of the palate which have separate innervations and yield strokes that actually trigger the flehmen response [12]. During flehmen, the attractants from the female enter the olfactory organ of the bull which, in turn, relays the signals to the hypothalamic areas of the brain [13]. The ‘flehmen behaviour show’ is the most definite and integral part of the premating scenario. Bioassays to quantify the pheromone activity have been designed and defined using the flehmen behaviour of bull [14]. In the present study, we have postulated that the flehmen behaviour of the buffalo bull in response to estrus (3 to þ1 day) and diestrus stages of female, by virtue of its ability to elicit the presence of estrus-indicating pheromones, could be used as a practical tool for detection of estrus. To our knowledge, a study of this nature has not been carried out in buffaloes so far.
2. Materials and methods The behavioural study was carried out in the Government Livestock Farm, Orathanadu, Tanjore District, South India. Twelve Bulls and twelve heifers (Bubalus bubalis) were used in the study. The animals were fed a standard diet and water was allowed ad libitum. Rectal examination of each heifer was performed regularly at one or two weeks intervals to verify the normal morphological changes in the internal organs. The flehmen behaviour was studied on the bulls under natural conditions of breeding during three consecutive estrous cycles (from 12 to þ12 days with day 0 estrus) including 36 estrous cycles in all. A total of 817 observations (180 estrus days and 637 diestrus days) was made during the experiment. The duration of each observation was approximately 15 min. During the observation period, the bull was allowed to investigate the female and the frequency of flehmen behaviour of each bull to each female was recorded throughout the estrous cycle. Generally, the bull sniffed the genital regions of the female and raised the neck, extended
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866
863
Fig. 1. Male displaying the flehmen behaviour after inhaling the pheromonal substance(s) from the vaginal region of female.
the chin and inhaled with slightly opened mouth, the tongue being held in a flat position and the upper lip curled so that the nostrils became partly closed (Fig. 1). The bull also manipulated the tongue during the flehmen behaviour. The statistical analysis was done by applying Student’s ‘t’ test on the data concerning the flehmen behaviour in estrus (3 to þ1) and diestrus [15].
3. Results The exhibition of flehmen behaviour by the buffalo bulls observed in the present study appears to be typical for buffalo reproduction. The flehmen behaviour was observed throughout the 36 estrous cycles as presented in Fig. 2. When the bull was released to investigate the female, he first inhaled the pheromonal substance(s) from the vaginal region by sniffing and elicited flehmen behaviour. When the bull identified the estrus specific compound(s) during the first flehmen behaviour, he continued smelling and displaying flehmen frequently known as ‘repeated flehmen behaviour’. If he did not find the specific compound(s) during investigation, he stopped with a single flehmen or without flehmen. In all, 817 observations consisting of 180 estrus (3 to þ1) and 637 diestrus days were made. The flehmen behaviour was exhibited in 806 comprising 365 in estrus and 441 in diestrus days. A total of 258 repeated flehmen behaviour was observed including a high frequency of 188 flehmen (72.9%) during estrus and 70 (27.1%) during diestrus. Average numbers of all (2:03 0:66) and repeated flehmen (1:05 0:64) behaviour during estrus periods were
864
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866 3.5
3
ALL FLEHMEN Flehmen numbers
2.5
REPEATED FLEHMEN
2
SIGNIFICANT ALL AND REPEATED FLEHMEN DURING ESTRUS PERIODS p<0.01 & p<0.001 RESPECT IVELY WHEN COMPARED WITH DIESTRUS
1.5
1
0.5
0
-12-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0
1 2
3 4 5 6 7
8 9 10 11 12
Days
Fig. 2. Occurrence of flehmen during estrous cycle (day ‘0’ estrus).
significantly higher than those of diestrus periods of all (0:69 0:25) and repeated flehmen (0:11 0:10). The statistical significance was higher (P < 0:001) in repeated flehmen behaviour of estrus as compared to that in diestrus. In case of all flehmen behaviour, the statistical significance was higher (P < 0:01) in estrus when compared to that in diestrus.
4. Discussion In mammals other than primates, males proceed to mate after receiving the specific signals from females. The females generally produce such signals from early estrus. The use of biological assay for simultaneous detection of estrus related odors seems to be a prerequisite for the chemical study of pheromones [14]. Flehmen is a unique behaviour shown by bulls and it could be used as a parameter for monitoring the estrous cycle [14]. It has been suggested that the flehmen behaviour takes place under the stimulation of the vomeronasal organ through which male ungulates discriminate estrus and non-estrus urine [16]. Interestingly, during the observation periods, the flehmen behaviour increased from day –4, peaked at day 0 (3:11 1:19) and decreased in late estrus. The present findings in buffaloes support the earlier reports in cows in that the flehmen behaviour is most frequent on the day of estrus and decreases during the diestrus phase [14]. Our observations on the flehmen behaviour of the buffalo bull to the females the day before estrus and throughout the estrus are similar to the report made on cows [17]. The present investigation clearly shows that estrus elicited significant repeated flehmen behaviour. Obviously, the occurrence of flehmen during estrus is due to the presence of specific pheromonal cue(s) present in the female urine/vaginal mucus. It is also interesting to note that even though diestrus elicited repeated flehmen behaviour (0:11 0:10, significantly lower P < 0:001 when compared to estrus), it may also be controlled by
S. Rajanarayanan, G. Archunan / Theriogenology 61 (2004) 861–866
865
certain sex pheromones. The presence of such pheromones, however, may be negligible and may have connection with some other events of estrous cycle, such as, interovulatory follicle growth [14]. The flehmen behaviour during the diestrus stages, nevertheless, had either singled or was non-responsive. It is well known that when bulls identify the specific pheromonal compound(s) they will continue to exhibit repeated flehmen and whenever such specific compound(s) are not identifiable, the bulls will stop with single flehmen [14,16]. Pheromonal compound(s) present in estrus provoke the manifestation of premating behaviour in the males. The males have the capability to discriminate between estrus and diestrus urine based on chemical cues present in it [18]. The flehmen is triggered with a high frequency through inhaling the estrus urine and sniffing the vaginal mucus. The sex pheromone believed to be present in the excretory products is released into the environment and perceived by the bull which eventually evokes the behavioural response. This is consistent with other studies reported in cows [14,19,20], sheep [21,22], cotton- top tamarins [5], equines [23] and elephants [24]. It is well known that the pheromonal compounds, particularly those involved in reproductive behaviour are mediated through the vomeronasal organ (VNO) which is situated in the nasal septum [25,26]. Therefore, it is also our future endeavor to find out the functional aspect of VNO in perception of chemical signal in relation to expression of flehmen in male buffaloes. The general concept of females to express their readiness for mating by producing specific olfactory signals is supported by the results of this study in the case of buffaloes. Since estrus specific chemical signal(s) is primarily involved in inducing the flehmen behaviour it can be concluded that specific pheromonal compound(s) present in urine/vaginal mucus are responsible for the flehmen behaviour and this can be used as a simple technique for detection of estrus in buffaloes. Further characterization of urinary/vaginal mucus pheromonal compound(s) in buffaloes is underway in our laboratory.
Acknowledgements We thank Dr. C. Natarajan formerly of the Indian Veterinary Research Institute, Izatnagar for his constant encouragement and critical reading of the manuscript. This investigation was supported by grants from the Department of Science and Technology, Government of India.
References [1] Keverne EB. Pheromones and sexual behaviour. In: Money J, Musaph H, editors. Handbook of sexology. North Holland: Elsevier Biomedical Press; 1977. p. 413–28. [2] Dominic CJ. Chemical communication in animals. J Sci Res (Banarus Hindu University) 1991;41:157–9. [3] Aron C. Mechanisms of control of the reproductive function by olfactory stimuli in female mammals. Physiol Behav 1979;59:229–84. [4] Schams D, Schallenberger E, Hoffman B, Karg H. The oestrus cycle of the cow: hormonal parameters and time relationships concerning oestrus, ovulation and electrical resistance of the vaginal mucus. Acta Endocrinol 1977;86:180–92.
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