OTU’s of 30 phyla, 59 classes, 124 orders, 215 families, 513 genera and 485 genera plus species were identified by the new generation sequencing. At the phylum level, a significant difference was found as the main effect for the factor of days prior to fixed-time artificial insemination (FTAI), for Actinobacteria (p = 0.03) and Fusobacterium (p = 0.03), in both cases these bacterial phyla are increased two days before FTAI in cows subjected to a protocol with a progesterone intravaginal device, compared to the group of cows 10 days before FTAI and just before the progesterone intravaginal device is inserted. No interaction was found between groups. Bacterial phyla with a higher relative abundance sequenced between Group I and II were very similar; Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The relative bacterial abundance obtained in Group III was Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria, and for Group IV, the relative bacterial abundance found was Proteobacteria, Firmicutes, Actinobacteria and Fusobacteria. Using the OTU's identified in each group, a heatmap and histogram for relative abundance for phyla were constructed utilizing OTU found in each group (Fig. 1).
Similarly, using the analysis method described, it was determined at the bacterial class´s level (Fig. 2). No significant interaction was found between the groups studied. In contrast, as the main effect in the postpartum groups evaluated, significant difference was found for the classes Deltaproteobacteria (p = 0.01), Alphaproteobacteria (p = 0.04), Campylobacteria (p = 0.04) and Anaerolineae (p = 0.05), in all these cases more OTU's of the bacterial classes were identified in cows between 50 and 100 days postpartum compared to cows with more than 101 days postpartum. Significant differences were also found as the main effect of the days prior to FTAI factor, as the classes Actinobacteria (p = 0.03) and Fusobacteria (p = 0.03) were increased in the group of cows used, two days before FTAI in beef cows subjected to a protocol with a progesterone intravaginal device. The relative abundance of the most relevant classes, found for groups I, II, III and IV was Clostridia, Bacteroidias, Gammaproteobacteria, Actinobacteria, Bacilli and Fusobacterias which in groups I and II do not appear, but which rises in relative abundance in groups III and IV.
In bacterial orders, interaction was found between the groups evaluated in the factorial design for the order Kinesporiales (p. 0.05) and Actinomycetales (p. 0.02). Significant differences were found between the postpartum days factor for Myxococcales (p. 0.02), Rhodobacterales (p. 0.03), Cellvibrionales (p. 0.04) and Campylobacterales (p. 0.04) where OTU's were significantly identified in cows between 50 and 100 days compared against those with more than 101 days postpartum and for the factor of days prior to fixed-time artificial insemination significant difference was found for orders; Micrococcales (p. 0.03), Pasteurellales (p. 0.03), Fusobacteriales (p. 0.03) where significant OTU's were determined two days prior to FTAI and Aeromonadales, where there was significance in cows 10 days prior to FTAI compared to cows 2 days before FTAI (p. 0.03),. Orders with the greatest relative abundance were Clostridiales, Bacteroidales, Bacillales and Micrococcales (Fig. 3).
As for the sequenced bacterial families, significant interaction was found for Kineosporiaceae (p = 0.02), Promicromonosporaceae (p = 0.03) and Actinomycetaceae (p = 0.05). In the postpartum group as the main effect, significant differences were found in the families Rhodobacteraceae (p = 0.03), Cellvibrionaceae (p = 0.04), Christensenellaceae (p = 0.04), Campylobacteraceae (p = 0.04) and Defluviitaleaceae (p = 0.05) where OTU's were significantly identified in cows between 50 and 100 days compared against those with more than 101 postpartum days. In the days leading up to FTAI, significant differences were found for the families Porphyromonadaceae (p = 0.03), Pasteurellaceae (p = 0.03), Micrococcaceae (p = 0.03), Fusobacteriaceae (p = 0.04) and Pseudomonadaceae (p = 0.05), families in which OTU's were sequenced significantly two days prior to FTAI compared to 10 days prior to FTAI just the day the protocol began, in addition, in the family Succinivibrionaceae OTU's were found significantly on day 10 prior to FTAI, compared to day 2 prior to FTAI (p = 0.02). The families with the highest relative abundance are Ruminococcaceae, Lachnospiraceae, Prevotellaceae and Fusobacteriaceae (Fig. 4).
No significant interaction was found between sequenced genus. In the postpartum group as the main effect, significant differences were found in the genera Atopostipes where OTU's were significantly sequenced in cows with less than 100 days postpartum (p = 0.02) unlike the Campylobacter (p = 0.04) and Negativibacillus (p = 0.05) where there was a significant difference in cows with more than 101 postpartum days compared against cows between 50 and 100 days postpartum. Similarly, significant difference was found for the genus Porphyromonas (p = 0.03), Glutamicibacter (p = 0.03), Fusobacterium (p = 0.04) and Pseudomonas (p = 0.05) where it is found were in greater abundance two days before FTAI compared to 10 days before FTAI, in contrast to the genus Ruminobacter where more OTU's were found on day ten prior to FTAI compared to day two prior to FTAI (p = 0.03). The genera with the greatest relative abundance they found are Fusobacterium, Acinetobacter, Roseburia and Bacillus (Fig. 5).
Among bacteria sequenced up to genus and species, significant difference in interaction was found for Arcanobacterium hippocoleae (p = 0.05) and Trueperella pyogenes (p = 0.05), additionally significant differences were found between postpartum groups as the main effect in Brachybacterium sacelli where more was found in cows between 50 and 100 postpartum days than in cows over 101 days postpartum (p = 0.05) and for the days leading up to FTAI as the main effect, significant differences were found for Serratia marcescens where more than 10 days prior to FTAI was found (p = 0.05) and Glutamicibacter creatinolyticus where more OTU's were sequenced on day 2 prior to FTAI (p. 0.05). The most commonly sequenced genus and species relative abundance are: Fusobacterium necrophorum, Histophilus somni, Moraxella Bovoculi and Glutamicibacter mysorens (Fig. 6)
Diversity by Shannon´s index
Diversity according to the Shannon´s index has a mean of 3.30 ± .71 SD for cows sampled on day − 10 of the protocol. The mean of Shannon´s index on day − 2 of the protocol is 2.54 ± .93 SD. There is a significant decrease (p = 0.006) between days − 10 and − 2. (Fig. 7)