Abstract
Background: The raw milk microbiota is closely associated with the physiology and pathology of the mammary gland, and plays a pivotal role in the development of offspring. The aim of this study was to examine the variability of raw milk microbiota diversity in relation to season, farm, parity, and subclinical mastitis status in Ningxia, China. Results: Raw milk samples were collected from 285 cows in Ningxia, China, followed by the exploration of microbiota using 16S rRNA high-throughput sequencing. Subsequently, all samples were categorized by season (summer vs. winter), health status (healthy vs. subclinical mastitis), farm origins (6 commercial dairy farms), and parity (primiparous vs. multiparous). Bacterial diversity, community composition, and abundance were assessed across these cohorts. Findings indicated significant variation in bacterial diversity of milk between samples collected from summer and winter. Summer samples exhibited higher bacterial richness compared to winter samples. Gut-related genera, including Parabacteroides, Staphylococcus, Corynebacterium_1, Sphingomonas, and Lactobacillus were found to be prevalent in summer milk samples. Conversely, Escherichia-Shigella, Pseudomonas, Streptococcus, Psychrobacter, Rhizobium, Bifidobacterium, and Clostridium_sensu_stricto_1 were commonly observed in winter samples. The subclinical mastitis cows showed a significantly higher alpha diversity value (Chao1 and Shannon indices) compared to the healthy cows (summer). Sporolactobacillus, Mycobacterium, Escherichia-Shigella, and Actionmycetaceae were identified as biomarkers in the subclinical mastitis cows. In addition, the top 20 KEGG pathways were significantly reduced in the subclinical mastitis group compared to the healthy cows in summer, particularly in membrane transport, cell motility, and signal transduction pathways. The bacterial diversity of milk varied significantly across farms. The bacterial composition was more similar between Farms A and B, and between Farms D and F. Whereas Farm C was distinct from all others. In addition, multiparous cows exhibited greater bacterial richness than primiparous cows. Conclusions: The study indicated that the milk microbiota composition varied with season, farm, health status, and parity. These findings provide insights into the raw milk microflora in this region and can guide local farms in optimizing management and quality control measures.