The present study demonstrated microbial diversity of Bos indicaus (Gir) cow during the estrous cycle under Indian conditions. Our study revealed a high diversity of bacteria and a limited diversity of Archaea. Cows from two farms were included in this report, although there was no difference in taxonomic analysis between the two farms (P=0.3516). This may be due to close proximity of farms and optimal feeding and management practices. There are reports (Quereda et al. 2020) on cow microbiota during the estrous cycle, however, are from herds of North America or Europe. In our study, we have analysed data from four different phases (D0, D4, D12, and D16) of estrous cycle, which are further investigated at stage level: follicular (D16 and D0) and luteal (D4 and D12). There are many factors which may influence the microbial diversity of vaginal microbiota including postpartum infections (Coleman et al. 1985).The microbiological diversity across the phase may be influenced by reproductive hormone concentrations presents during the estrous cycle (Mahesh, et al. 2020, Mahesh, et al. 2021). Progesterone, on the other hand, has been reported to colonise the vaginal microbiome and enhance bacterial diversity. Similarly, our study found that hormonal shifts had a considerable impact on microbial diversity during the four estrous phases. According to (Quereda, et al. 2020), the vaginal microbiota of cows was statistically different at the follicular and luteal stages of the estrous cycle in dairy heifers, which supports our findings that the follicular stage of the estrous cycle has much more variety than the luteal stage. (Fig. 2B). Finally, except for the diestrus phase, the principle coordinate plot (Fig. 7) demonstrates a greater commonality in microbial diversity. In this analysis, there is a substantial difference in alpha and beta diversity between the Luteal and Follicular stages. No significant phyla were observed, however, Micrococcus, Stenotrophomonas, UGC-010, Massilia, Methylobacillus, and Pseudomonas genus was observed statistically significant in the Luteal and follicular stages. Only the diestrus phase of the luteal stage reveals a statistically crucial distinction in the microbiota abundance. However, Lactobacillus is the most abundant Firmicutes genus in women vaginal microbiota, this genus is not abundant in the other mammalian species (Mehta et al. 2020, Ravel et al. 2011). Although in other mammals, lactic acid bacteria does not dominate as vaginal microbiota (Reid et al. 1985, White et al. 1989).
Our results on levels of progesterone hormone during estrous cycle demonstrated significantly lower level during D0 then D12 phase which may affect the microbial diversity in the vaginal environment. As diversity analysis also indicates that D12 phase shows maximum diversity among the all stages of estrous cycle which can be influenced by the progesterone level. Also, alpha diversity and Beta diversity statistical data suggest the same. Though shift of principal microorganisms occurs during the bovine's ovarian cycle (Miller et al. 2017). However, the most mentioned genera of bovine’s vagina LAB are Lactobacillus, Pediococcus, Leuconostoc, and Weissella, which are phylogenetically close to each other (Miller et al 2017) and usually measured together as the Lactobacillus group. Women's vaginal microbiota mainly consist of Lactobacillus genus (Mehta, et al. 2020, Swartz et al. 2014), however, bovine shows a low abundance of Lactobacillus genus (Mahesh et al. 2020, Mahesh et al. 2021), and shows high diversity of the phyla Proteobacteria and Fermicutes. Fig. S1 depicts bacterial changes in the lactobacillae family during the four phases of the estrous cycle. While Lactobaillus bacteria are found in the cow's vaginal tract, they can help to prevent or treat urogenital infections (Cribby et al. 2008). Quereda et al. (2020) observed that Lactobacillus is not dominant in the microflora of cow heifers which supports the results of our study. Swartz et al. (2014) presented that the most profuse genera were Streptobacillus spp. and Aggregatibacter spp. were detected in 90% of cows and 80% of ewe samples. In our study, there are four phyla Bacteroidota, Firmicutes, Proteobacteria, and Verrucomicrobiota which are statistically significant after statistical correction by the Benjamini-Hochberg FDR test. This was in relation to previously findings on the core bovine uterine microbiota and postpartum vaginal microbiota (Laguardia-Nascimento et al. 2015). Firmicutes and Proteobacteria consist of 94% of the phyla of the vaginal diversity during the estrous cycle. Swartz et al. (2014) presented those Ewes and cow were predominantly colonized by the Proteobacteria, Bacteroidetes, and Fusobacteria. So many reports are supporting the results of our study. Giannattasio-Ferraz et al. (2020) they also demonstrated that most superabundant phyla are Firmicutes that contain 40-50% of the diversity followed by Proteobacteria, Bacteroides, and Actinobacteria in vaginal microflora. Differences in bacterial diversity were observed between these studies because of breed, feed, environmental condition, geological location, age, etc. A common vaginal microbiota composition among breeds of Bos taurus indicus (Gyr and Nellore), cattle phyla predominant by Firmicutes (~40–50%), Bacteroidetes (~15–25%), and Proteobacteria (~5–25%; Carmina et al. 2014). Ault et al. (2019) discovered that Verrucomicrobiota is abundant in postpartum cows, with a higher percent abundance in cows that were pregnant after insemination compared to cows who did not become pregnant. Similarly, they also observed that there are 4 families and 17 genera showed relative abundance >1%. f_Leptotrichiaceae, f_Corynebacteriaceae, Ruminococcaceae UCG-005, Mycoplasma, Helcococcus, Bacteroides, Campylobacter, Porphyromonas, Histophilus, Ureaplasma, Rikenellaceae RC9 gut group, f_Lachnospiraceae, Streptococcus, Alistipes, Facklamia and Coprostanoligenes group were the most abundant families and genera observed. Similar genera were discovered in lower abundance in the findings of Quereda et al. (2020), which validates our findings. In our study, we observed only two significant genera and two significant families. The only significant genera and family were Bacillus, Alcaligenes, f_Enterobacteriaceae, and f_Morganellacea. Micrococcus. Stenotrophomonas, UGC-010, Massilia, Methylobacillus genus was observed statistically significant at luteal stage and Pseudomonas genus in the follicular stage.
Composition of the bacterial communities studied here were highly heterogeneous between different phases of the estrous cycle and their anatomical and physiological differences may influence microbiota and reproductive tract (Laguardia-Nascimento, et al. 2015). Unlike some authors' hypothesis that hormonal maturity has a significant impact on the development of vaginal microbiota in cattle, the results obtained in this study demonstrate that hormonal maturity seems to have significant impact on the establishment of vaginal microbiota in cattle.