Our previous study showed differences in the CVM bacterial community with respect to various phases of the oestrous cycle (Srinivasan et al. 2019), in which the Firmicutes phylum was more abundant during the oestrus phase. Probably, expression of certain specific bacterial genes is higher during estrus phase than the other phases. The present investigation revealed expression of specific gene(s) in S. pasteuri during the estrus phase. The available information indicates that the CVM bacterial strains get influenced during estrus in buffalo. It has also been reported that the vaginal bacterial population is affected by the cyclical variation during the oestrous cycle (Otero et al. 1999; Otero et al. 2000).
In the present study, S. pasteuri were incidentally observed in buffalo CVM during estrus. Similar studies have been reported in various animals. Association of vaginal Simonsiella spp in reference to estrus phase has been documented in lions and leopards (Callealta et al. 2018). It is interesting to note that Streptococcus and Staphylococcus are the most dominant bacteria in healthy cows during the reproductive cycle (Amin et al. 1996). Staphylococcus (70%) and Escherichia (15%) were observed more frequently during natural and induced estrus of ewes (Orihuela et al. 2019) and all the isolates had a high sensitivity to ciprofloxacin antimicrobials that evaluated vaginitis regulation (Mohammed et al. 2017). The present finding supports the previous report of the presence of Staphylococcus pasteuri, which comes under the phylum Firmicutes, and is abundant during estrus (Srinivasan et al. 2019).
In addition, bacteria such as Bacillus spp., Staphylococcus spp., and Streptococcus spp., have been identified in vagina of ewe (Manes et al. 2010) as well as cows (Otero et al. 1999; Otero et al. 2000; Zambrano-Nava et al. 2011). These reports match with the present finding of bacterial community identified in CVM of buffalo. On the other hand, major bacterial genera in the vagina of Gyr and Nellore breed cows include Aeribacillus, Bacillus, Clostridium, Bacteroides and Ruminococcus (Giannattasio-Ferraz et al. 2019). The present analysis also depicted the presence of Bacillus spp. in buffalo exclusively during preestrus and diestrus phases. Notably, Lactobacilli were found to be very low in bovine vaginal microbiota which was confirmed by culture-dependent and culture-independent methods (Otero et al. 2000; Quereda et al. 2020; Srinivasan et al. 2019). The present finding reveals the absence of Lactobacillus sp. in buffalo CVM. Overall, the results obtained in the present study are consistent with previous reports.
The mice system was used to screen the volatile compound samples. Among 7 samples tested with mice, S. pasteuri secretory substance exhibited to be more attractive than the other cultures. The results are consistent with an earlier report that mice are capable of discriminating the estrus and non-estrus samples. A recent study provides supportive evidence that mice are highly sensitive to volatiles produced by the microbes (Peixoto et al. 2018). Truly, the mice study helped to pick out the bacteria potentially responsible for volatiles production.
GC-MS analysis indicated six volatiles, acetic acid, propanoic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid, as produced by S. pasteuri. It is known that short-chain fatty acids such as acetic-, propanoic- and butyric acid, are relatively abundant due to bacterial fermentation in colonic lumen which regulate entero-pathogenic colonization by host immunomodulation in human (Smith et al. 2013; Wrigley 2004). A recent study shows that acetic, valeric, caproic and myristoleic acids are at significantly higher levels in the milk of estrus cow (Zebari et al. 2019). The fatty acids, acetic and propanoic, have been identified in the faeces of cow, and varied in relation to the time of ovulation (Mozūraitis et al. 2017). The present investigation supports the previous reports of presence of acetic, propanoic and valeric acids as produced by CVM bacteria in estrus buffalo.
Since estrus-specific S. pasteuri produces volatiles, we focused on testing the role of S. pasteuri secretory substance in bull behaviours which confirmed the estrus-specificity and its use as a reliable estrus indicator. Interestingly, bull exhibited reproductive behaviours such as flehmen and mounting, which are important in assessing sexual desire of bull as response to S. pasteuri secretory substance. The present study also corroborates previous findings that the estrus-specific faeces pheromone has a significant effect on flehmen and mounting behaviour in buffalo (Karthikeyan et al. 2013). It has been reported that bulls exhibit this reproductive behavioural patterns in response to the presence of a mixture of volatiles in the CVM of estrus cow (Karthikeyan and Archunan 2013; Rajanarayanan and Archunan 2004; Sankar and Archunan 2004). Vaginal pheromones have been reported to be acetic acid, propanoic acid, and 1-iodoundane, in cows (Sankar and Archunan 2004) and oleic acid as an estrus indicator in buffalo (Karthikeyan and Archunan 2013) to stimulate flehmen and mounting behaviour response in bulls. The present findings suggest that the volatile compounds of bacterial origin may act as attractants of the bull and induce the olfactory system to elicit flehmen and mounting behaviours in order to maintain the health and management during reproduction. Since S. pasteuri is capable of secreting the volatiles viz., acetic, propanoic, isobutyric, butyric, isovaleric, and valeric acids, they can be considered as major bacterial volatiles that induce the buffalo bull’s sexual behaviour. For instance, acetic acid, 2-butanone and oleic acid have been shown to improve Zebu bull sexual behaviour and total sperm production (Mondal et al., 2019). Thus, this study reports the presence in the buffalo vaginal fluid of volatile compounds secreted by CVM bacteria which facilitate the expression of male reproductive behaviours, which in turn suggests that these volatiles are putative pheromone compounds produced during oestrus in buffalo.
Further studies are needed to find the mechanisms associated with the specific effects of S. pasteuri volatiles. Characterization of protein profile of S. pasteuri from buffalo CVM during estrus is required to understand the host-bacterial interaction and their role in chemo-signal communication that would enlighten the reproduction management in buffaloes.