Introduction

It is a major challenge of the bull handler at any semen collection station to provide maximum sexual arousal prior to semen collection. It helps to harvest a maximum number of semen doses from any elite bulls. Zebu bulls are shy in nature shows sexual sluggishness as compare to exotic breed. Proper utilization of the most effective methods of sexual preparation and stimulation of bulls in breeding bull stations can result in mounting, ejaculation, optimal spermatozoal harvests and maintenance of bull’s libido. It is a big challenge to explore optimum potentiality of zebu bulls during semen collection at semen station without giving exposure to natural visual and tactile cues. The handling of bulls is also important for quality and quantitative semen production. Bull handlers give maximum focus on visual stimulation like false mount or change of teaser animal if bull shows unwillingness to mount or certain exercise around the dummy etc. but less attention is given on the tactile stimulation like snipping, licking, flehmen response, tongue manipulation and rump batting etc. It is very much common that both the visual and tactile stimulation are playing important to the breeding bulls for exhibiting of sexual behaviour in artificial semen collection centre. In case of Zebu bulls, they are used to go for tactile stimulation and take time for semen donation compare to exotic breeding bulls during artificial semen collection. Cattle bulls exhibit sexual behaviours in presence of different estrus specific body fluids like faeces (Sankar and Archunan, 2008), urine (Archunan and Kumar, 2012), saliva (Sankar et al., 2007), milk (Sankar and Archunan, 2004) and vaginal fluid (Sankar and Archunan, 2011). It was evident that olfactory cues under artificial condition stimulate male sexual behaviours but applications of estrus specific body fluids are not explored fully. Nowadays, different estrus specific molecules were identified from different estrus body fluids and applied for improvement of sexual behaviours of cattle breeding bulls (Sankar and Archunan, 2008; Archunan and Kumar, 2012; Le Danvic et al., 2015). The molecules like coumarin, squalene, 1, 2-dicholroethylene, 6-amino-undecane, oleic acid, and 2-butanone were reported by Le Danvic et al. (2015) in estrus urine of Holstein Friesian cows; acetic acid & propionic acid were also found in faeces (Sankar and Archunan, 2008) of exotic cattle; whereas 1-iodoundecane was found in faeces (Sankar and Archunan, 2008) and urine (Archunan and Kumar, 2012) of Bos taurus cattle during estrus phase. The application of estrus molecule or mixture of different molecules showed longer flehmen and increase in the number of mounts (Sankar and Archunan, 2008; Sankar et al., 2007). Scanty information is available to explore the efficacy of the molecules to enhance the sexual behaviour of indigenous bulls. Therefore, the present work was planned to observe different behaviours of restrained bulls towards estrus specific molecules under shed before semen collection.

Materials and Methods

The study was conducted on ten Sahiwal breeding bulls maintained at Artificial Breeding Research Centre, ICAR-NDRI, Karnal, Haryana. The bulls were maintained under loose housing system and uniform management system. The ten animals were exposed to the glycerol-water solution (50:50, v: v) first as control and then exposed to estrus specific molecules in the switch over design. The estrus specific molecules like squalene, 1-iodoundecane, acetic acid, coumarin, propionic acid, oleic acid and 2-butanone were purchased from Sigma-Aldrich Company, USA and the molecules were solubilized individually in a non-pressurized aerosol dispenser as 1.0 % solution in glycerol-water solution (50/50, v:v) and used as treatment. All solutions were prepared fresh at morning before application and 2-3 ml of solution was sprayed on both the nostril after 20-30 minutes of washing the bulls. The separate aerosol dispensers were used for an individual molecule. The behavioural activities i.e. Flehmen, tongue rolling, licking, penile movement, making a sound, tail rising, urination, defecation, arch back condition and back kicking of bulls were observed for a period of 15-20 minutes. Behavioral score as 10 point scale was made by sum off total behavioral activities. The single point was given as ‘one’ for each sexual behaviour expression otherwise zero (0) point in case of there was no behavioral expression.

Statistical Analyses

The behavioral score as well as individual behaviors (Flehmen, tongue rolling, licking, penile movement, making a sound, tail rising, urination, defecation, arch back condition and back kicking) were compared between molecules and control. The data were analysed in JMP^® 12.2.0 Software by oneway ANOVA, followed by means comparisons for all pairs using Tukey-Kramer HSD test.

Result and Discussion

Sexual behaviour score of sahiwal breeding bulls after exposure of estrus specific molecules was presented in Table 1 and Fig. 1.

Table 1: Sexual behaviors score (Mean ± SEM) of Sahiwal breeding bulls (n=40) after exposure of estrus specific molecules

Estrus specific molecules are volatile in nature as a result exact amount of compounds released during estrus phase is very difficult to detect. In one of the study, Rajanarayanan and Archunan (2011) used 1 % concentration of estrus specific molecules to observe the reproductive behaviour; therefore we also used 1% concentration for the molecules. The present findings revealed that control, Squalene, 1-iodoundecane, Acetic acid, Coumarin, Propionic acid, Oleic acid and 2- butanone showed a behavioural score of 2.17±0.06, 3.55±0.16, 4.92±0.13, 7.45±0.18, 4.10±0.10, 4.07±0.11, 6.42±0.16, 7.52±0.21, respectively.

Fig 1: Sexual behaviours score after exposure of different estrus specific molecules

Bulls showed maximum behaviour score after exposure of acetic acid, followed by 2- butanone and oleic acid. Bulls showed lowest behaviour score in case of control followed by squalene. Similar to our observation, Le Danvic et al. (2015) reported that 2-butanone and oleic acid enhanced libido of bulls as compared to coumarin, squalene and 1, 2-dichloroethylene. In a study, Archunan and Kumar, (2012) reported 1-iodoundecane acted as a strong stimulator for sexual activities than di-n-propyl phthalate in the bulls. The variation in the results with respective molecules may be due to various molecules, variation in the breed, exposure duration of molecules, the way of application and concentration level of molecules. It is also evident from the literature that estrus specific molecules are different in different breeds, species and different phases of estrous cycle (Sankar and Archunan, 2008; Le Danvic et al., 2015; Rajanarayanan and Archunan, 2011). In Estrus urine, coumarin, squalene, 1, 2-dicholroethylene, 6-amino-undecane, oleic acid, and 2-butanone were found by Le Danvic et al. (2015). The 1- iodoundecane found in Jersey cows in estrus (Kumar et al., 2000) displays a similar chemical structure to the 6-amino-undecane but it was specific to the pre ovulatory stage, identified by Le Danvic et al., 2015. So, there was chemical composition variation was evident in different breeds and various estrus specific body fluids (Le Danvic et al., 2015). The molecules like acetic acid & propionic acid were also found in vaginal fluid (Sankar and Archunan, 2011) and saliva (Sankar et al., 2007) of cattle, whereas, 1-iodoundecane was found in urine (Archunan and Kumar, 2012) during estrus phase. The less effective role of coumarin in the present study may be due to the fact that coumarin is naturally present in many plants and secreted in urine, besides that coumarin degrade in the liver and its degraded products are excreted through urine (Felter et al., 2006) and type of diet (Le Danvic et al., 2015). Among the seven molecules, acetic acid, 2- butanone and oleic acid were most effective for better sexual preparation of Sahiwal bulls as several behavioural responses were exhibited better after exposure of the molecules. The molecules were identified in exotic breeds and crossbred cattle. Therefore, there is need to explore the estrus specific molecules present in estrus specific body fluid of indigenous cattle.

In the case of licking and the tongue rolling, there were no such significant variation seen when all the individual molecules compared with control solution and quite common behaviour among the individual animal that might be due to natural habit to take a sense of any unknown fluid. The maximum Flehmen response was observed with acetic acid (0.87±0.05) and 2- butanone (0.85±0.06) compared to control solution. Tail raising, penile movement and making sound were found higher in acetic acid (0.55±0.08, 1.00±0.00 and 0.38±0.08) and 2- butanone (0.65±0.08, 1.00±0.00 and 0.30±0.07). In other individual behaviours like arch back condition, back kicking, urination and defecation were also observed more in percentage with acetic acid and 2-butanone compared to control (Table 2).

Table 2: Various sexual behaviour expression (proportion) in Sahiwal breeding bulls exposed to different estrus specific molecules

Means bearing different superscripts within a column as A, B differ significantly at p <0.01

In similar line, application of various estrus body fluids and serum to free bulls showed several behavioral activities like attraction, hyper salivation, vocalisation, head butting, body blocking, Mounting etc. and in case of restrained bulls, head direction to the sample, smell the sample, salivation, licking of the sample, tongue manipulation, flehmen, penis protrusion and dribbling from penis (Rivard and Klemm, 1989) was observed. Archunan and Kumar, (2012) observed attraction, Sniffing, Salivation, Urination, Tongue Manipulation, Flehmen, Vocalization and Penis Protrusion after applying of estrus specific molecules in the bull pen. In our study, tail rising, defecation, arch back condition were seen because after the nasal spray the bulls were kept for 15-20 minutes for behavioral observation under shed and that time bulls were excited to express the sex drive. It may be due to stimulation via the hypothalamic system that generates pulses of gonadotropin-releasing hormone (Rekwot et al., 2001). Therefore, olfactory cue is one of the important factors playing role in arousing sexuality or libido. Similar to our observation, all the molecules did not act in similar way when applied in front of the bulls or applied in nostril showed various behaviours like sniffing, licking, flehmen reaction etc. (Archunan and Kumar, 2012; Le Danvic et al., 2015).

Conclusion

In Sahiwal bulls, acetic acid and 2-butanone are the suitable estrus specific molecule for sexual stimulation before semen collection. The molecules may be effective in uniform sexual preparation to optimize the semen production performance of Sahiwal bulls. There is need to identify estrus specific molecules in secretion of body fluids of Indigenous cow to improve the pheromone efficiency for better sexual preparation of indigenous bulls.

Acknowledgement

The author wishes to sincerely thank ICAR-NDRI, Karnal for providing the Junior Research Programme to the first author and Director ICAR-NDRI for providing the necessary facilities and fund for the work.

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