Variation in the Milk Microbiota of Healthy Dairy Cows Across Parity and Lactation Stage

To characterize variation in the milk microbiota of clinically healthy dairy cows, milk samples were collected repeatedly from four cows throughout lactation and analyzed by 16S rRNA gene amplicon sequencing. Alpha-diversity analysis showed no significant effects of parity, lactation stage, udder quarter position, or the interaction between lactation stage and position. Although sample-level PERMANOVA indicated significant associations of beta diversity with cow identity and parity, these associations were not supported when the repeated-measures structure was appropriately accounted for. Restricted-permutation PERMANOVA showed no significant effects of lactation stage, udder quarter position, or their interaction, and exact permutation testing at the individual-cow level showed no significant effect of parity. Ordination and hierarchical clustering analyzes nevertheless revealed substantial variation in bacterial community composition among individual samples. Hierarchical clustering based on genus-level relative abundance separated most samples into two major groups characterized by relatively high and low diversity. The high-diversity group showed a relatively high abundance of the class Clostridia, whereas the low-diversity group was characterized by a predominance of Gammaproteobacteria, particularly Escherichia/Shigella in some samples. Overall, the milk microbiota of healthy dairy cows showed substantial sample-to-sample variation, but the present data did not provide clear evidence that parity, lactation stage, or udder quarter position systematically influenced microbial diversity. These findings provide baseline information on variation in the milk microbiota of healthy dairy cows and highlight the importance of accounting for repeated measurements and individual variation in studies of the bovine milk microbiota.