APPLICATION OF CATTLE PHEROMONES IN THE ANIMAL REPRODUCTION & MANAGEMENT

Pheromones are types of chemicals that are released by organisms as a means of communication with organisms of the same species. Some animals release it through their urine, their skin or even in their feces, and it is detected using smell. Pheromones can be released for a variety of reasons including readiness to mate, an alert to danger, showing a certain territory or even when an animal is ovulating!

The word pheromone comes from the Greek word pherein which means to carry or to bear. This makes sense because pheromones carry or bear information. There are two types of pheromones: releaser and primer.

Releaser pheromones get an immediate response. Have you ever seen an ant wandering around and wondered, ‘How in the world is this little thing going to find her way home?’ Ants release releaser pheromones, which are detected as a smell to other ants, when they have found food. This lets the other ants know that they can return to the nest in order to feast. Once the food runs out, the ants release a separate pheromone that lets the ants know that the food is gone.

Another ant example of a releaser pheromone, is an alarm pheromone. In this case, the ants that are in danger release a pheromone that basically says ‘come over and help me!’ This results in ants coming to their aide.

Primer pheromones, on the other hand, do not cause an immediate response. Let’s look at an example:

When female rats are housed together without a male, their reproductive cycle stops or is slowed. This is signaled through pheromones released in the female rats’ urine. Since there is no male around, there is no point in expending the excess energy.

Another example in primer pheromones in rats is used for sexual availability. When a female is ready to mate, she releases a pheromone that excites male rats and even causes them to mount her .Chemical signals play a major role in mammalian reproduction and behavior. While the existence of sexual pheromones and their significance in reproductive behavior are very well known in laboratory mammals, there is little information about the species-specific chemosignal in cattle. However, in the last decade there have been some important and relevant reports for cattle pheromones. This chapter provides a comprehensive account on cattle pheromones and their role in reproduction and social behaviors. Sexual attraction, mother-young interactions, estrus indication, estrus induction, puberty acceleration, reducing the postpartum anestrus, hormonal stimulation, and enhancing the penis erection are some of the classical events influenced by pheromones. During the reproductive cycle of cattle, the male exhibits a specific behavior of flehmen that is used as an indicator to detect estrus. Furthermore, the sensory system involved in the perception of pheromones is mediated through the vomeronasal organ (VNO) and main olfactory system (MOS). The sex attractant volatile compounds have been identified in the urine of the cow, buffalo, and horse. The urinary volatile compounds identified in female buffalo are capable of enhancing the sperm quantity, which can be considered as a significant finding for cattle pheromones. The pig appeasing pheromone (PAP) is characterized from the mother skin used to reduce the fighting behavior among the piglets whereas the boar salivary pheromone is found to induce puberty attainment and boar mate. Similarly, PAP is also present in the horse, cat, dog, and human and plays a significant role. In the goat and sheep, the volatiles identified in the ram and buck wool fleece to stimulate LH and ovulation induction. There is plenty of scope for the application of cattle pheromones in the animal reproduction and management.

The pheromone signals are known to have a potential role in animal reproduction and management (Archunan 2009; Buck 2000; Dominic 1991; Rekwot et al. 2001; Tirindelli et al. 2009). The sources of chemosignals are urine, feces, vaginal secretions, saliva, and specialized scent glands including the odor produced from hair and wool (Albone 1984; Aron 1979; Patra et al. 2012; Van den Hurk 2007). Communication of the timing of the physiological event of ovulation and coordination of sexual behavior are important for successful fertilization (Schams et al. 1977; Ziegler et al. 1993); the success rate of artificial insemination in cattle mainly depends on the time of estrus it is inseminated. If the female fails to conceive when it is inseminated in the nonestrus period, it is a great economic loss estimated around $300 million to the dairy industry in the United States and in India the approximate loss would be several crores. The detection of estrus and diagnosing early pregnancy are the major problems in farm animals, particularly in buffalo. The other problems like irregular or prolonging of the estrous cycle, anestrus, fighting behavior among young ones, mother-young bond, unmotivated males, and poor farm animal management including stress are considered as major issues in farm animals (Rekwot et al. 2001) that need to be addressed. Moreover, pheromones have not been exploited for the purpose of enhancing livestock production.

The animal releases volatile odors into the surrounding atmosphere, most of which are waste products of metabolism in which emission of some compounds closely related to reproductive activities are termed as chemical signals (Hradecky 1975). The estrus-related odors are present only during the preestrus and estrus stages. These chemical signals have been reported to be volatile and nonvolatile molecules that are perceived through the main or accessory olfactory system (Brennan and Keverne 1997; Tirindelli et al. 1998). In mammals, structurally and anatomically, the olfactory systems are classified into two types: the MOS and the accessory olfactory system (AOS), specialized for the detection and transmission of pheromonal information (Halpern and Marinez-Marcos 2003; Mucignat-Caretta et al. 2012).

In the last two decades, there has been a considerable increase in the knowledge of the chemistry of pheromones in cattle (Rajanarayanan and Archunan 2011; Rameshkumar et al. 2000; Sankar and Archunan 2004; Sankar et al. 2007), pig (Signoret 1970), horse (Kimura 2001), goat (Delgadillo et al. 2006), and sheep (Gelez and Fabre-Nys 2006). These findings suggest that cattle pheromones may act together for influencing precopulatory behavior and successful reproduction. It further indicates that cattle pheromones may be a single compound or a mixture of compounds and that each of the major fractions was faithfully involved in conveying specific signals related to reproductive and social behaviors. The pheromones have not been exploited as much as they can for farmer utility although they have a tremendous role in reproduction. The present review provides insights about the nature of the compounds, behavioral characterization, and practical utility of cattle pheromones that are available

Cattle reproduction is vital for successful management of farm animals. In the reproductive cycle estrus has a central role in cattle reproduction. The period of the estrous cycle of cattle is calculated from one estrus (heat or phase of sexual receptivity) to the next estrus. The average estrous cycle periods are 21 days in bovine and buffalo and 17 days in ewes (Hall et al. 1959; Tilbrook and Cameron 1989). The estrous cycle is subdivided into four phases based on the dominant hormone or ovarian structure and reproductive behavior during each phase. The stages of the bovine cycle are preestrus, estrous, metestrus, and diestrus. The days are calculated as follows: day 0 is considered to be estrus, days 1–5 are metestrus, days 6–17 are diestrus, and days 18–20 are preestrus (James and Ireland 1980).

Generally, the estrous cycle of cattle is divided into two phases: (1) the folicular phase and (2) the luteal phase. During the follicular phase, under the influence of FSH the follicle starts to develop and undergoes ovulation; when the LH surge occurs the estrogen level is also high. The luteal phase begins when the corpus luteum (CL) is formed about 5 to 6 days after the cow is in heat and ends when the CL regresses from about 17 to 19 days of the cycle. Progesterone levels are high during this phase of the cycle and estrogen levels are low (De Jarnette et al. 2009). The season is also reported to influence the length of the estrous cycle in bovine (Sankar and Archunan 2012).

The female expressed estrus symptoms include vaginal swelling, mucus discharge, bellowing, frequent urination, and mounting on other animals during heat (estrus) period (Karthikeyan 2011; Rajanarayanan 2005; Rajanarayanan and Archunan 2004; Rameshkumar 2000). In order to attain optimum reproductive performance in dairy farms it is essential that herdsmen be aware of signs of estrus and their reliability, and then only they can accurately select females for artificial insemination, the most crucial aspect of successful conception being the timing of the insemination. Maximum conception rates are obtained when the insemination is done from 8 to 12 hours after the onset of estrus (also known as standing heat). Hence, detection of estrus is very much essential for achieving success in artificial insemination service.

The role and importance of chemical signals in reproductive behavior has been well studied in several species of mammals (Archunan 2009; Balakrishnan and Alexander 1985; Dominic 1991; Rasmussen et al. 1996; Rekwot et al. 2001). One of the striking examples is that the male identifies the estrus through pheromonal signals released from the female. The most likely sources of such signals are urine and cervical mucus secretion (Archunan 2009). For example, in cattle, bulls can detect pheromone odors and differentiate between estrus and nonestrus urine (Sambraus and Waring 1975).

The ultimate criterion for effective pheromonal communication of the sexual status of a female is a clear message emitted from her body fluids (Rivard and Klemm 1989) during the estrus period by which the male is being attracted, followed by mating (Hradecky et al. 1983; Klemm et al. 1987). When a male is brought to a female in estrus the male generally sniffs the genital region of the female and exhibits several behaviors. One such significant behavior is the retraction of the upper lip, wrinkling of the nose, and baring gums. This is always accompanied by deep breathing and may also include the elevation and extension of the head. This specific behavior was observed by German scientist and postulated the term “flehmen” (Hradecky et al. 1983). The main function of flehmen has been hypothesized to be the discrimination of chemical signals in females (Bland and Jubilan 1987; Rajanarayanan and Archunan 2004; Sankar and Archunan 2004). Pheromonal compounds present in the body fluids during estrus periods provoke the manifestation of premating behaviors in the males including flehmen. 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 (Vandenbergh 1999). The repeated flehmen behavior show is the most definite and integral part of the premating scenario. This unique behavior response has been reported in the bovine (Dehnhard et al. 1991; Hradecky et al. 1983; Rajanarayanan and Archunan 2004; Sankar and Archunan 2004), sheep (Blissitt et al. 1994), and horse (Ma and Klemm 1997). Hence, based on the available reports flehmen behavior can be considered as one of the best indicators for the female in estrus.

Bulls exhibited flehmen behavior following the perception of a cow’s body fluids like urine (Archunan and Rameshkumar 2012; Rameshkumar 2000), saliva (Sankar et al. 2007), feces (Sankar and Archunan 2008), and milk (Sankar and Archunan 2004) of the estrus stage. Our study demonstrated that the bull exhibited flehmen behavior when exposed to dummy cows sprayed with estrus urine (p < 0.001) as compared to that of nonestrus urine, and suggested that estrus urine was capable of inducing the flehmen behavior in the bull (Archunan and Rameshkumar 2012). The chemosensory responses and premating behavior of adult males to the female vaginal fluid have been well demonstrated in B. taurus (Klemm et al. 1987; Paleologou 1979; Sankar and Archunan 2011).

Under field conditions, visual stimuli probably play a role in stimulating male sexual behavior, but in vitro tests with cervicovaginal mucus clearly show that there is an olfactory component, independent of vision, in the stimulation of male reproductive behaviors, such as sniffing, licking, and repeated flehmen in response to vaginal chemical cues (Hradecky et al. 1983; Sankar and Archunan 2004, 2011). The flehmen behavior is the prime response against the olfactory signals of an estrus female leading to other behavioral responses. Based on this observation, it is strongly believed that there is a correlation between flehmen and other behavioral responses to olfactory signals of estrus females.

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 ovulation without any behavioral signs of estrus (Dobson and Kamonpatana 1986), thereby making the detection of heat in buffaloes more difficult than in cows (Danell et al. 1984).

The problem of estrus detection in the buffalo is overcome by using the herd bulls that monitor olfactory and gustatory cues routinely (Reinhardt 1983; Williamson et al. 1972). Bulls receive the estrus-specific chemical signal from females and then exhibit flehmen behavior. This is preceded by fast tongue strokes over the rostral and medial part of the palate that have separate innervations and yield strokes that actually trigger the flehmen behavior (Jacobs et al. 1980). During flehmen, the attractants from the female enter the olfactory organ of the bull which, in turn, relay the signals to the hypothalamic areas of the brain (Vandenbergh 1999).

Rajanarayanan and Archunan (2004) reported that flehmen behavior increased from day 4 (preestrus period), peaked at day 0 (estrus), and decreased in late estrus. Further, the bulls exhibit a significant repeated flehmen behavior when exposed to estrus females. It is also interesting to note that even though the diestrus phase elicited the flehmen behavior, it is significantly lower (p < 0.001) when compared to estrus. From this report it was concluded that flehmen behavior is controlled by certain sex pheromones. The presence of such pheromones, however, may be negligible and may have a connection with some other events of the estrous cycle, such as interovulatory follicle growth (Hradecky et al. 1983). The flehmen behavior during the diestrus stage, nevertheless, had either singled or was nonresponsive. It is well known that when bulls identify the specific pheromonal compound(s) they continue to exhibit repeated flehmen and whenever such specific compound(s) are not identifiable, the bulls will stop with a single flehmen (Estes 1972; Hradecky et al. 1983). We carried out a detailed study on flehmen behavior with regard to urinary compounds and obtained an interesting result. The flehmen behavior of the bull was greatly influenced by the estrus-specific compound as compared to that of diestrus compound (Karthikeyan and Archunan 2013; Rajanarayanan and Archunan 2011). The finding clearly indicates that estrus-specific compound is closely associated with the expression of flehmen behavior. Figure shows the exhibition of flehmen by male buffaloes as soon as inhaling the estrus urine.

Flehmen behavior is also reported in several ungulates other than the cow and buffalo. Bland and Jubilan (1987) reported ram flehmen after a ram sniffed urine voided by the female. Flehmen in response to urine was exhibited least often in the presence of the estrus ewe due to the low occurrence of marking behavior by the female at this time. Flehmen also occurred after the ram investigated the vulva of the ewe, most frequently on the day before estrus. These observations support the hypothesis that the occurrence of flehmen by the ram is due to an olfactory mechanism for confirming the reproductive state of the ewe. The pheromone detected by flehmen appears to be produced in the vagina and carried in the urine.

The flehmen response is found to appear in the male goat with regard to the female sexual interest (Ladewig et al. 1980). The horse exhibited flehmen behavior by drawing back their lips in a manner that makes them appear to be grimacing or smirking. In the horse, a stallion showed a significant response of flehmen behavior toward an estrus mare more than to a nonestrus mare (Marinier et al. 1988). Furthermore, the occurrence of sniffing and flehmen was used to determine the discriminating ability of the stallion and it was found that stallions are capable of discriminating the sex of a horse by the feces source (Stahlbaum and Houpt 1989). In the case of the pig, boars exhibit flehmen sometimes simultaneously toward sows; however, this is the only ungulate that did not show flehmen normally (Dagg and Taub 1970).

It has been believed for a long time that the two chemosensory systems—the MOS and the vomeronasal system (VNS)—are responsible for the perception of odorants in mammals. The MOS is considered to be responsible for recognizing the conventional volatile odorant molecules, whereas the VNS is thought to be tuned for sensing pheromones. Recent studies have demonstrated that both chemosensory systems, together with additional olfactory organs, are involved in pheromone detection (Halpern 1987; Mucignat-Caretta et al. 2012; Tirindelli et al. 2009).

Jacobson originally described the vomeronasal organ based mainly on domesticated animals (cat, cow, dog, goat, horse, pig, and sheep); he also described the organ in other mammals such as the tiger, camel, buffalo, deer, and seal (Doving and Trotier 1998). The presence of this organ has been confirmed in most mammals including marsupials (Wohrmann-Repenning 1984). VNO is situated in the vicinity of the second cheek tooth. The mean length of the VNO, measured from the Papilla incisiva to the caudal end of the vomeronasal cartilage (VNC), was 4 mm for minks, 15 mm for cats, 50 mm for dogs and pigs, 150 mm for cows, and 200 mm for horses. The VNC characteristics did not appear to be affected by age in any of the species studied (Salazar et al. 1995). It was observed that the VNO from its tip, where incisive openings were located backward a pear shape cartilaginous capsule encloses the organ, but this capsule is incomplete (Abbasi 2007). Figure 16.2 shows the location of VNO in the buffalo. Animals demonstrate different behavior patterns that are adaptations to investigating odorant sources. Some of these behavior patterns are certainly related to the investigation of the odorants, while others have to do with the entry of odorants into the VNO (Doving and Trotier 1998).

Experimental study showed that when the VNO in guinea pigs was impaired the males failed to mount. Females with impaired organs did not show lordosis, lost interest in their partner, and seldom became pregnant (Gerall 1963). Peripheral dedifferentiation of the VNS produces severe sexual behavior deficits in both male and female hamsters (Powers and Winans 1975; Winans and Powers 1977). In the goat, the posterior part of the VNO contains sensory epithelium that facilitates the nonvolatile molecule from the oral cavity to the VNO for the flehmen response (Ladewig et al. 1980). The formation of mother-young bond between ewes and lambs is reported to be mediated through olfactory cues and the VNO is primarily involved for the neonatal recognition in sheep (Booth and Katz 2000). However, another study was performed on both the olfactory systems to test the pheromone odor in enhancing LH secretion in ewes. The VNO was ablated in ewes and then exposed male fleece; it was found that there is no difference in the LH secretion in ewes by exposing to the male odor. By contrast, destruction of the main olfactory epithelium by zinc sulfate irrigation greatly reduced the LH secretion in ewes while exposing the male fleece (Gelez et al. 2004); this indicates that VNO is not necessary for the perception of male odor in sheep. Further experimental evidence showed that ablation of VNO in the sow did not alter the attraction and standing posture when exposed to boar saliva; however, the role of the MOS in this aspect was not tested (Dorries et al. 1991). As far as the cow and buffalo are concerned, the importance of both the main and vomeronasal systems has not been tested so far in the context of chemical communication. Flehmen behavior is believed to be involved in the transport of fluid-borne chemical stimuli, such as sex pheromones, from the oral cavity to the VNO. During flehmen behavior, the intermittent nostril licking apparently delivers the stimulus material to the VNOs via the nasal route, possibly compensating for reduced oral access. The VNS is reported to be involved in several pheromonal effects with regard to puberty acceleration, pregnancy block, induction of estrus, and mating behavior in rodents (Gelez et al. 2004). However, recent reports strongly indicate that the MOE was involved primarily in the attraction from a distance, while the VNO played a major role in close proximity (precopulatory behavior), indicating that the olfactory–vomeronasal systems play a synergistic role in the detection of estrus and the mating process (Achiraman et al. 2010). Even though the above study was done in the mouse, a similar synergistic role of the olfactory–vomeronasal systems would be possible in other mammals, including ungulates.

Pheromones in the urine, feces, or from cutaneous glands can be perceived through the olfactory system to elicit both behavioral and endocrine responses in conspecifics, and biostimulation (priming pheromones) can exert profound effects on reproductive activity via the hypothalamic system (Dulac and Wagner 2006; Rekwot et al. 2001). The role of pheromones has been shown in several reproductive events such as sexual maturity, induction of ovulation, reduction of postpartum anestrus, and coitus in many mammalian species including rodents, wild animals, feral populations, swine, sheep, goat, and cattle (Burns and Spitzer 1992).

The exposure to boars induced puberty at an earlier age than gilts reared without being exposed to a boar and reduced the postpartum period in lactating sows indicates the potential role of primer pheromone (Brooks and Cole 1970; Kirkwood et al. 1981). The “ram effect” (primer pheromone) has been reported to accelerate the onset of estrus activity and promote varying degrees of estrus synchronization (Schinckel 1954). It is important to note that the ewes of most breeds are anestrus for some portion of the year and exposure of ram to anestrus ewes reduces the length of the anestrus period leading to the return of estrus activity (Rekwot et al. 2001).

Biostimulation is an inexpensive and suitable method for extensive management systems and it is an excellent example of the potential underlying “control systems technologies aimed at controlling reproductive performance” (Martin 1995; Signoret 1970). The role of primer pheromones in cattle reproduction is not as clearly defined as that in other species such as sheep, goats, and swine, possibly due to nutritional and other environmental stresses (Roberson et al. 1991). It is important to note that the presence of a teaser bull did not hasten puberty or alter the size of ovaries in a group of prepubertal heifers (Berardinelli et al. 1978; Macmillan et al. 1979; Roberson et al. 1987).

One of the main problems in cattle reproduction is anestrus, defined as absence of the estrous cycle, which has to be decreased in order to obtain a more sustainable production in the livestock industries (Fiol and Ungerfield 2012). Biostimulation (male or bull effect) can be defined as the stimulus provoked by the presence of males, which induces estrus and ovulation through genital stimulation, pheromones, or other external cues (Chenoweth 1983). The stimulus provoked by the introduction of the males can act through different pathways, including olfactory, visual, and auditory signals (Ungerfield 2007). In cattle, males’ excretory products and cervical mucus from estrus females enhance ovarian function, both in postpartum cows (Berardinelli and Joshi 2005; Wright et al. 1992) and prepubertal heifers (Izard and Vandenbergh 1982).

Berardinelli and Joshi (2005) evaluated resumption of cyclic activity in postpartum, anovular, primiparous cows exposed to bulls or the excretory products of bulls. The cows were allowed in the pens along with a male for 12 hours daily during 70 days of experiments. The authors found that anestrus postpartum length was greatly reduced when the female was exposed to males or the excretory products of males, suggesting that the biostimulatory role of bulls appears to be mediated by pheromone present in their excretory product. The nature of the compound present in the excretory product is not yet identified (Berardinelli and Tauck 2007; Fernandez et al. 1993). Burns and Spitzer (1992) observed a similar reduction in postpartum interval to first estrus in cows exposed to bulls or to androgenized females. The effects of male exposure on cyclic activity have been evaluated mainly in Bos taurus taurus and other possible effects of biostimulation in Zebu (Bos taurus indicus), both in the postpartum cow (Rekwot et al. 2001; Soto Belloso et al. 1997), and prepubertal heifer (Rekwot et al. 2001). The biostimulation was also found effective to reduce the anestrus period in female buffaloes, Bubalus bubalis (Gokuldas et al. 2010; Ingawale and Dhobe 2004).

Improving reproductive efficiency in beef and dairy cattle should be one of the main objectives to obtain a more sustainable production. In this context, sociosexual stimulus, like biostimulation, represents low-cost and hormone-free alternatives when used alone or in conjunction with other techniques to increase reproductive results.

In ungulates, bulls routinely investigate the urine of anogenital areas of females presumably to determine their reproductive state. The involvement of chemosignals in the cow’s reproductive process is well documented; the female produces a specific odor during estrus in urine that is perceived by the bull (Archunan and Rameshkumar 2012; Denhard and Claus 1996; Rameshkumar et al. 2000). The dispersion of estrus-specific compounds in the cow’s body fluids has been demonstrated previously in swabs from the fluids (vaginal secretion, urine, milk, and blood) (Kiddy and Mitchell 1984; Rivard and Klemm 1989). The presence of estrus-specific volatiles was confirmed in milk, but the study did not find any compounds that were qualitatively different between stages but found there were significant quantitative differences in 15 compounds in milk (Weidong et al. 1997).

Klemm et al. (1987) found that blood acetaldehyde levels decreased rapidly just before, or at the onset of estrus, and suggested that estrus and ovulation could potentially be predicted by monitoring the levels of acetaldehyde in milk, saliva, sweat, or breath. Acetaldehyde was also found to be estrus-specific in bovine vaginal secretions (Weidong et al. 1997); however, it has been tested in a bull bioassay previously (Presicce et al. 1993) and was not behaviorally active when tested as a singular component.

Several studies have focused on vaginal secretions found during estrus. Preti (1984) patented a method for detecting bovine estrus based on the quantification of methyl heptanol in vaginal secretion. Another finding showed that the concentration of free fatty acids in estrus vaginal discharge increased gradually before estrus and decreased rapidly thereafter (Hradecky 1986). By contrast, the concentration of free fatty acids in urine, but not in vaginal discharge, was affected by the luminal concentration. Klemm et al. (1987) found nine estrus-specific compounds in urine that were tested positively in a bull behavioral assay. These compounds included two ketones, four amines, one alcohol, one diol, and one ether. Sankar and Archunan (2004) reported that other body fluids such as vaginal fluid, saliva, feces, and milk collected from estrus period contained chemical cues that attract the opposite sex.

Bulls can discriminate estrus from nonestrus urine and estrus urine has been shown to elicit sexual behavior in the cow. Rameshkumar et al. (2000) identified two estrus-specific compounds (i.e., 1-iodo undecane and di-n-propyl phthalate), however, the behavioral assay later clearly indicated that the 1-iodo undecane is capable of stimulating the bull response for coitus and hence was considered as a biochemical marker for bovine (Archunan and Rameshkumar 2012).

Sankar and Archunan (2004) studied the behavioral assay among the proestrus, estrus, and diestrus samples; the test clearly showed that the estrus vaginal fluid was found to be more variable (p < 0.001) than the saliva, feces, and milk. The vaginal secretions induced the maximum response in bulls showing flehmen behavior, thereby confirming that the vaginal secretions from the estrus cow contained pheromone(s), which along with urinary pheromone(s) result in attracting the bull.

Furthermore, our laboratory study reported that 11 different volatiles are identified in cow saliva from three different reproductive phases (Sankar et al. 2007). Among the identified compounds, the following compounds, trimethyl amine, acetic acid, phenol 4-propyl, pentonic acid, and propionic acid were specific to estrus. The behavioral assay has confirmed that the compound from a salivary source (i.e, trimethylamine) may be involved in attracting the males. Interestingly, the chemical profiles of estrus feces differed significantly from other phases by specific substance such as acetic acid, propionic acid, and 1-iodo undecane (Sankar and Archunan 2008); the behavioral assay also confirmed that these three compounds elicit a series of reproductive behaviors in the bull. It is evident that LH pulse frequency was greatly enhanced in the heifer by exposure to vaginal mucus and urine collected from estrus cows (Nordeus et al. 2012); however, the chemicals responsible for influencing LH alteration are not known. The above investigation on cow pheromone and behavioral characterization have convincingly showed that urine, feces, vaginal secretion, saliva, and milk appear to be prominent sources for cow pheromone production for estrus indication and influence several reproductive behaviors including hormonal stimulation.

Another interesting aspect is to detect estrus by interspecific communication. The ability of dogs (Hawk et al. 1984), rats (Denhard and Claus 1988), and mice (Rameshkumar et al. 2008; Sankar and Archunan 2005) to detect estrus-specific odor in cow urine is reported. Trained dogs can distinguish the cow luteal phase (Hawk et al. 1984; Kiddy and Mitchell 1984). In addition, a detailed experimental study was carried out using the mouse as an experimental model to evaluate which of the body fluids of the estrus cow has more attracting ability towards mice (Sankar and Archunan 2004). The test clearly showed that the estrus vaginal fluid was found to be the most attractive source followed by saliva, feces, and milk. The findings suggest that the specific odors present in the estrus phase not only attract the conspecific but also attract between species.

The occurrence of silent ovulation without any behavioral symptoms of estrus is the major problem in buffalo reproduction (Danell et al. 1984; Dobson and Kamonpatana 1986). Unlike in the cow and other ungulates, visual signs of estrus are not prominent in the buffalo, which make it difficult to detect heat effectively. Hence, there is a need for a reliable method to detect estrus in the buffalo. Recent reports show that the buffalo exhibits a reproductive behavior and releases chemosignals from few sources that can be exploited for estrus detection (Archunan 2009). Rajanarayanan and Archunan (2004) demonstrated that the average number of all flehmen behavior (2.03 ± 0.66) and repeated flehmen (1.05 ± 0.64) behavior in the bull upon exposure to an estrus heifer were significantly higher than those of diestrus periods of all (0.69 ± 0.25) and repeated flehmen (0.11 ± 0.10).

The nature of the volatiles present in the urine is confirmed and proved to be a pheromonal signal (Rajanarayanan and Archunan 2011). Fourteen different volatile compounds from all the stages of the estrous cycle were identified in urine samples, which included phenol, ketone, alkane, alcohol, amide, acid, and aldehyde. Amongst the identified compounds, a few, such as 2-octanone, 2-methyl-N-phenyl-2 propenamide, decanoic acid, N,N-bis (2 hydroxy ethyl) dodecanamide, tetradecanoic acid, and hexadecanoic acid, were commonly found throughout the cycle (preestrus, estrus, postestrus, and diestrus). However, the volatile compounds 1-chlorooctane, 4-methyl phenol, and 9-octadecenoic acid occurred only during estrus. Behavioral assay further revealed that bulls were attracted and exhibited repeated flehmen behavior toward 4-methyl phenol, whereas bulls exhibited penis erection and mounting response by exposure to 9-octadecenoic acid. By contrast, the other compound 1-chlorooctane did not show any such sexual behavior in bulls. It is interesting to note that these compounds are absent during postestrus and diestrus, which indicate that these compounds are characteristic to the estrus period. The reports convincingly conclude that 4-methyl phenol acts as a sex attractant compound and 9-octadecenoic acid acts as mounting response. It is remarkable to note that the urinary sex pheromones have been shown to enhance the sperm production after exposing to the nasal region; this has been awarded a patent (Archunan and Rajanarayanan 2010).

The nature of the volatile compounds produced during estrus in buffalo has been predicted. The existence of compounds phenol, 3-propyl phenol, and 9-octadecenal in preestrus urine suggests that these three volatiles may be considered as the preindicators for estrus. It is also interesting to note that the volatile phenol present in preestrus probably becomes 4-methyl phenol in estrus urine by the addition of a methyl group. Similarly, 9-octadecenoic acid, which appears in estrus, is probably derived from the preestrus compound of 9-octadecenal. Presumably, a change occurs in the formation of chemical compounds from the preestrus to estrus phase.

In addition to sex pheromone characterization in urine, other body fluids such as feces, vaginal mucus, and saliva have evinced great interest in view of identification of the volatile compounds and their behavioral assay, which may be considered as another important source for sex pheromone production. A specific volatile compound, 9-octadecenoic acid, has been identified as estrus-specific in vaginal mucus (Karthikeyan and Archunan 2013); it is to be remembered that the volatile compound 9-octadecenoic acid is already identified in estrus urine of the buffalo (Rajanarayanan and Archunan 2011). In feces, two volatile compounds, 4-methyl phenol and trans-verbenol, have been identified and confirmed as estrus indicator, suggesting that feces may be considered as a source of estrus indicator in the buffalo (Karthikeyan et al. 2013). The 4-methyl phenol (P-cresol), a unique volatile compound, can be considered as a common compound of estrus-specific since it is present in urine (Rajanarayanan and Archunan 2011), feces (Karthikeyan et al. 2013), and saliva (Karthikeyan 2011). Hence, this compound might be produced under the influence of estrus hormones.

As far as the boar pheromone is concerned, three types of pheromones have been recognized (1) boar mate pheromone, (2) puberty accelerating pheromone, and (3) appeasing pheromone. Chemical communication in the pig is notably mediated by saliva. The boar mate pheromone produced from pig saliva against the estrus sow is remarkably involved in the organization of the sequence of sexual behavior to attract the estrus females and facilitate the display of a receptive posture (Signoret 1970). The steroid pheromones such as 5α-androsterone and 3α-androsterone induce the appropriate standing response for mating and these odors are essential for a prolonged act of coitus in pigs. An aerosol boar mate is now marketed to aid pig artificial insemination practice by inducing the immobilization reflex in the estrus female (Booth 1984). The puberty accelerating pheromone is also released from saliva in the male pig, which is reported to induce early puberty in young female piglets (Pageat and Teyssier 1998). The boar pheromones have been shown to accelerate puberty in gilts by about 30 days to synchronize estrus and to reduce the postpartum period in lactating sows (Brooks and Cole 1970). Furthermore, those gilts reached early puberty by the presence of a boar odor, have higher ovulation rates, regular estrous cycles, and improved reproductive potential than the controls (Izard 1983).

Appeasing pheromone is another important chemical communication involved between mother and young pups of pigs. Generally, after weaning, the piglets will fight when group-housed, which leads to reduce greatly the feeding among piglets. The fighting behavior is a major concern among the farmers because the pigs do not gain weight due to underfeeding. Odors isolated from the skin of milking sows have been shown to reduce agonistic behavior in piglets (Pageat and Teyssier 1998). The putative maternal pheromone is composed of six fatty acids in different proportions (Pageat 2001): hexadecanoic acid, cis-9-octa decenic acid, 9,12-octyladecanoic acid, dodecanoic acid, tetradecanoic acid, and decanoic acid. The commercial synthetic analog had similar effects when tested in industrial husbandry (McGlone and Anderson 2002). This pheromone can be considered as an outstanding discovery in the mammalian pheromones.

The small ruminants such as the goat and sheep are economically important farm animals worldwide for their milk, meat, and wool. The response of the female goat to the male is weak but can be induced by the introduction of teaser bucks that had previously been exposed to long days (Delgadillo et al. 2006; Rivas-Munoz et al. 2007). The odor of the male and its sexual behavior plays a primary role in inducing ovulation, while vocalizations appear to facilitate the display of the does’ estrus (Delgadillo et al. 2006). Additionally, the sebaceous gland has been indicated as sources of primer pheromone production in goats (Iwata et al. 2000; Sugiyama et al. 1981). The primer pheromones produced by the male have been known to stimulate the reproductive neuroendocrine system of anestrus animals (Chemineau 1987; Knight and Lynch 1980). Exposure of a male into a group of anestrus females leads to the activation of sexual activity by influencing the LH secretion and synchronization of ovulation in the sheep and goat (Knight et al. 1978; Ungerfeld et al. 2004). Just as the influence of primer pheromones in the acceleration of puberty in rodents is well established, the chemosginals from the ram and buck have been found to accelerate puberty in the sheep and goat (Shelton 1978; Underwood et al. 1944). The evidence indicates that 4-ethyl octanoic acid (4EOA) identified in major fleece does not evoke an LH response in a female conspecific but its derivatives might have pheromonal activity (Iwata et al. 2003). It was also found that the synthetic 4EOA possessed a releaser pheromone activity that induced specific behavior in the recipient, and female goats showed some interest in the odor (Iwata et al. 2003; Sasada et al. 1983).

The pheromones secreted through the ewe’s wool and wax (Tilbrook and Cameron 1989) and vaginal secretions (Ungerfeld and Silva 2004) sexually attract the ram; however, the nature of the pheromonal compound present in the female is not yet investigated. The volatile substance(s) responsible for female LH secretion has been found in male sheep hair, skin, and their extracts obtained by organic solvents (Cohen-Tannoudji et al. 1994). It is further reported that the compounds 1, 2-hexadecanediol and 1, 2-octadecanediol appear to be responsible for this pheromonal effect in sheep. The synthetic compounds are reported to be effective in stimulating LH release in anestrus ewes. In the domestic sheep, primer pheromones are considered the most important signals involved in sociosexual stimulation of the reproductive processes and male-female interactions induce changes in the pulsatile rhythm of the LH secretion in both sexes, which influences reproductive endocrinology.

The occurrence of olfactory memory for the formation of mother-young bond between ewes and lambs is another interesting aspect of a milestone in support of the involvement of pheromonal communication. Sheep have been used as the best model to study the mother-young relationship (Bouissou 1968; Kendrick et al. 1992; Nowak et al. 2011). The strong bond between ewe and lamb formed shortly after parturition particularly within few hours is very crucial and critical for lamb survival (Keller et al. 2005; Porter et al. 1991). The onset of maternal responsiveness and the development of mother-young attachment in sheep are under the control of hormonal and sensory stimulation (Kendrick et al. 1992; Keverne et al. 1993; Nowak et al. 2011). Offspring recognition through olfaction in most females is generally believed to be based on the offsprings’ individual olfactory signature (Poindron et al. 1988; Porter et al. 1991), which is believed to originate from its body coat (Alexander and Stevens 1982) from the anal region (Alexander et al. 1983). It is reported that majority of the odors from the anal region in animals have been associated with pheromones (Bean 1982). The first day of life is entirely dependent on the success of the first suckling episodes (Nowak et al. 2011). If suckling is prevented during the first few hours after birth by covering the ewes udder, the development of a preference for mother is lowered (Nowak et al. 1997), suggesting that sensory outputs from emanating the suckling influence the development of filial attachment.

Amniotic fluid is an important substance in sheep for the establishment of maternal responsiveness toward neonates, facilitating their initial acceptance by the mother (Poindron et al. 2010). Amniotic fluid also carries individual olfactory cues from the neonates that help the formation of the maternal bond. Recently, several volatile organic compounds from lamb wool have been identified; these compounds serve as olfactory cues for neonatal recognition (Burger et al. 2011). However, the exact chemosignals involved in the maternal responsiveness toward the lamb are yet to be determined. It is known that the cell proliferation and survival in the adult brain are influenced by several internal and external factors (Lledo et al. 2006); for instance, the physiological status of pregnancy and parturition has been found to regulate cell proliferation in rodents. Regarding the establishment of maternal selectivity, it is hypothesized that downregulation of cell proliferation occurs in specific areas of the sheep brain and as well as in the main olfactory bulb during the early postpartum period (Brus et al. 2010; Levy et al. 1990). This could facilitate the development of olfactory perpetual memory that is retained in favor of the survival of the newborn neurons that might somehow assist in the learning process.

The role of biostimulation in the horse is very much needed in the context of horse breeding; however, the research on chemical communication in the horse is very much limited. Mares come into estrus several times a year (i.e., horses are a seasonal polyestrus species). A mare is sexually receptive towards a stallion for 5–7 days and ovulation occurs in the final 24–48 hours of estrus (Kiley-Worthington 1987). The precise determination of the estrus period is difficult and special qualification is needed to effectively elucidate the proper time of insemination (Mozuraitis et al. 2012). A mare signals estrus by urinating in the presence of a stallion, raising her tail, and revealing the vulva. A stallion approaching with a high head will usually nudge and nip a mare, as well as sniff her urine to determine her readiness for mating (Kiley-Worthington 1987).

Ma and Klemm (1997) detected 45 urinary volatile compounds at the estrus and diestrus stage of mares. Mozuraitis et al. (2012) analyzed the estrus urine samples and found 150 urinary volatiles; among the compounds, m- and p-cresols occurred significantly in greater amounts in estrus when compared to nonestrus. A great increase in amounts of p-cresol in urine samples from all mares of different breeds during the most active stallion acceptance periods provides a good signal to stallion that a mare is in estrus; thus p-cresol might be consider as a sex pheromone component. It is further reported that p-cresol is able to influence the penile erection in horse (Buda et al. 2012). In addition to urinary sources, sex pheromones such as palmitic and miristic acid are identified in feces and found to show the estrus signal to attract a stallion (Kimura 2001). The finding indicates that sex pheromones are produced from more than one source as it is already recorded in the cow and buffalo.

Odorant binding proteins (OBPs) are the soluble proteins of the lipocalin superfamily found in the mucus layer lining the olfactory epithelium of mammals (Mitchell et al. 2011; Pelosi 1994). The OBP is almost of similar structural sequence to the pheromone carrier protein that is present in almost all the sources of pheromone production in mammals (urine, saliva, sweat, vaginal mucus, etc.), and the prime function of the pheromone carrier protein is to bind the pheromone compounds and release them to the environment for manifesting the effect. OBPs are the first in the relay mechanism in pheromonal chemical reception because they provide the link between the chemical signal present in the environment and the odorant receptors located in the olfactory and peripheral sensory systems in mammals (Pelosi 1994). Major functions have been suggested for OBPs, such as scavenging odorants (Vincent et al. 2004) and protecting the airways (Mitchell et al. 2011). The OBP is well documented in bovine mucosa (Pelosi 1994) and the 1-octen-3-ol has been found to be the natural ligand of bovine OBP. Since 1-octen-3-ol is an important component of bovine breath and a very potent attractant for many insects, this indicates that the role of bovine OBP may be to clear the breath of 1-octen-3-ol to reduce the attraction of insects (Ramoni et al. 2002).

In the buffalo, OBP is reported in saliva; this OBP undergoes posttranslational modification (Rajkumar et al. 2010) and it is presumed that it may bind with the salivary pheromone molecule 4-methyl phenol. In addition, OBP has been found in the nasal mucus of the buffalo, suggesting that OBP may bind the odorants for further processing. In the pig, the nasal mucosal OBP has been reported and it has a specific role to bind with the steroid pheromone molecule (Scaloni et al. 2001). Available evidence on OBP in vertebrates concludes that OBP plays a pivotal role in the perception of the odorant/pheromones, contributing to all process. Furthermore, the previous investigation suggests that the binding and metabolic activity of the urine odorant 5α-androstan-3-one involved the odorant binding process in sheep olfactory mucosa (Krishna et al. 1995).

The potential role of pheromones has been updated in several aspects so as to enhance cattle reproduction and livestock management. Based on the large number of data obtained so far in cattle pheromone, the following applications are derived:

• 1. Puberty acceleration by male pheromone. Advancement of puberty is one of the important reproductive events that has been well established in cattle. The male pheromone is capable to advance the puberty in females than the attainment of normal puberty in several species of mammals including sheep, goat, pig, and cow (Rekwot et al. 2001). This enhancement of puberty in young cattle is considered to have a potential role in cattle production.

• 2. Estrus synchronization and estrus induction in anestrus by male pheromone. Introduction of a male into a group of anestrus females during the nonbreeding season results in the activation of LH secretion and synchronization of ovulation in the sheep and goat. This event is generally called the male effect and is widely used in husbandry of these species.

Reference-On Request