Effects of Ketosis on Milk Composition in Dairy Cows.
In the cows with ketosis, blood BHBA concentrations, with an average of 3.03 ± 0.22 mmol/L, were significantly higher, compared with the values in the healthy cows (Fig. 1A). The milk SCC in all of the ketosis cows were higher than 500,000 cells/mL, with an average of 464.3 ± 152.3 cells/mL (Fig. 1B). In addition, milk components analysis showed that except the proportion of milk protein remained unchanged, the percentage of lactose decreased significantly, while milk fat, urea nitrogen, as well as the ratio of milk fat to protein increased significantly in ketosis cows than healthy cows (Fig. 1C-G).
Microbiota 16S rDNA Amplicon Sequencing in Rumen Fluid and Milk in Dairy Cows.
We analyzed 36 samples by 16S rDNA amplicon sequencing, including 10 rumen fluid samples and 10 milk samples from ketosis cows, 8 rumen fluid samples and 8 milk samples from healthy cows. Overall, 8,372 OTUs were identified from the bacterial 16S rDNA profile in rumen fluid. At the phylum and genus level, a total of 72 phyla and 1026 genera were observed. A total of 10,623 OTUs were identified from the bacterial 16S rDNA profile in milk. At the phylum and genus level, a total of 89 phyla and 1193 genera were observed. To confirm adequate sequencing depth for these analyses, we generated the species accumulation boxplot for all of the samples and found our coverage to be sufficient for further analyses (Additional file 1).
Changes of Bacterial Community in Rumen Fluid and Milk between Ketosis and Healthy Cows.
The bacterial richness in rumen fluid and milk was estimated by Chao 1 and ACE index. Combined with Wilcox rank sum test, the richness of rumen microflora in ketosis cows was significantly lower than in healthy cows, while the richness of milk microflora in mastitis cows was significantly higher than in healthy cows (Fig. 2A-B). The bacterial diversity in rumen fluid and milk was estimated by Shannon and Simpson index. The result showed that there was no significant difference in rumen fluid and milk between ketosis and healthy cows (Fig. 2C-D). PCoA of the weighted UniFrac metric revealed a shift of community diversity among different groups along principal coordinates PC1 and PC2 (Fig. 3A). NMDS ordination performed on the Bray-Curtis dissimilarity showed that the bacterial community profiles of rumen fluid in ketosis cows was clear dissimilar from healthy cows (Fig. 3B).
Rumen Fluid and Milk Microflora at Phylum and Genus levels between Healthy and Ketosis Cows.
The relative abundance of the main bacterial taxa at different taxonomic levels in rumen fluid between ketosis cows and healthy cows were shown in Additional file 2. Both rumen and milk bacterial community were dominated by the following 5 bacterial phyla: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteriota and unidentified_Bacteria (Fig. 4A), accounting for more than 90%. However, the most abundant bacterial taxa in rumen microbiota were Firmicutes and Bacteroidetes. The ratio of Firmicutes to Bacteroidetes has traditionally been considered as a biomarker for metabolic potential of the gut microbiota[23]. We found that the Firmicutes to Bacteroidetes ratio significantly increased in ketosis cows than healthy cows(1.41 in KR vs 0.96 in HR)(Fig. 4B). At the genus level, the predominant genera in rumen fluid were Prevotella, Aeromonas, Succiniclasticum, Luteimonas, Thermomonas, Christensenellaceae_R-7_group, Ruminococcus, Rikenellaceae_RC9_gut_group, NK4A214_group and Kocuria, while in milk were Streptococcus, Vibrio, Shewanella, Prevotella, Staphylococcus, Lactobacillus, Coxiella, Escherichia-Shigella, Acinetobacter and Ruminococcus (Fig. 4C-D). Among the ruminal bacterial community, the single most abundant genus was Prevotella and its abundance significantly decreased in ketosis cows. The microbiota of milk samples was dominated by the genus Streptococcus.
Differential Bacteria in Rumen Fluid and Milk between Ketosis and Healthy Cows.
The distribution of common and unique OTUs among different samples is illustrated by Venn diagrams. In rumen fluid samples, there were 3154 OTUs uniquely present in healthy cows, 899 OTUs uniquely present in ketosis cows and 3564 OTUs shared between the ketosis and healthy cows (Fig. 5A). In milk samples, there were 1549 OTUs uniquely present in healthy cows, 2626 OTUs uniquely present in ketosis cows and 5856 OTUs shared between the ketosis and healthy cows (Fig. 5B). Moreover, Venn diagram showed that a total of 4608 OTUs shared by the rumen and milk samples in healthy cows, while 2957 OTUs shared by the rumen and milk samples in ketosis cows (Fig. 5C-D). MetaStat analysis showed that in the rumen fluid microbiota, at genus level, the relative abundance of Prevotella, Ruminococcus, Succinivibrionaceae_UCG-001 and Streptococcus were significantly decreased in ketosis cows, while the relative abundance of Luteimonas, Thermomonas, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, NK4A214_group, Paracoccus, Acetitomaculum, Prevotellaceae_UCG-003, Deinococcus, Saccharofermentans, Butyrivibrio and other low abundance genus were significantly increased in ketosis cows (Fig. 6A). LEfSe analysis was used to find biomarker in ketosis cows. The results showed that F082, Luteimonas, Thermomonas, Christensenellaceae_R-7_group, Rikenellaceae and Lachnospiraceae were enriched in rumen fluid from ketosis cows, while Prevotella was enriched in rumen fluid from healthy cows. (Fig. 6B-C).
Furthermore, LEfSe analysis revealed that Staphylococcus and F082 were enriched in milk from ketosis cows, while Corynebacterium, UCG-005, Acinetobacter and Coxiella were enriched in milk from healthy cows(Fig. 7A-B). Interestingly, we found that both F082, an unclassified bacterial family assigned to Bacteroidales, and Thermomonas were enriched in milk and rumen fluid from ketosis cows. In addition, MetaStat analysis revealed that the abundance of Acinetobacter and Oscillospiraceae_UCG-005 in milk were reduced simultaneously with those in rumen in ketosis cows. However, in ketosis cows, the abundance of Corynebacterium was decreased significantly in milk, while it was increased significantly in rumen (Fig. 7C).
Correlation Analysis of Rumen Microflora with Blood BHBA and Milk Components.
Correlation between the blood BHBA and milk components and bacterial taxa with top 35 relative abundance at genus level were illustrated in a heatmap (Fig. 8). The abundance of Luteimonas, Thermomonas, Christensenellaceae_R-7_group, Kocuria, Paracoccus, Corynebacterium, Acetitomaculum and Deinococcus showed significantly positive correlation with blood BHBA concentration. However, the abundance of Prevotella, Ruminococcus, Succinivibrionaceae_UCG-001 and Streptococcus correlated negatively with blood BHBA concentration, as did the lower taxa Erysipelotrichaceae_UCG-002, Syntrophococcus, Lactobacillus and Dialister. Highly consistent with the MetaStat analysis, we observed a higher abundance of the bacteria positively correlated with blood BHBA in ketosis cows than in healthy cows, while we observed a lower abundance of the bacteria negatively correlated with blood BHBA in ketosis cows than in healthy cows (Fig. 6A). Interestingly, most of these bacterial genera significantly related to blood BHBA are related to the SCC in milk, and their correlation are consistent. Moreover, the milk fat, milk urea nitrogen increased with BHBA at the same time, so did the same correlation with these bacteria. In contrast, Prevotella, Ruminococcus, Succinivibrionaceae_UCG-001 and Streptococcus showed significantly positive correlations with milk lactose.