关键词: 四君子散, 瘤胃发酵, 挥发性脂肪酸, 瘤胃微生物, 16S rRNA高通量测序

Abstract:

The objective of this study was to explore how Sijunzi San influences the rumen microbial community and fermentation processes in postpartum dairy cows, to investigate the potential mechanisms of its spleen-invigorating effects. Sixteen multiparous Holstein cows with similar body weight (600 kg±30 kg) and body condition score (3.0-3.5) were randomly assigned to either an experimental or a control group. The experimental group received an oral administration of 300 grams of Sijunzi San following calving, whereas the control group was given an equal volume of clean drinking water, both for a duration of 7 consecutive days. On the eighth day, rumen fluid was collected from both groups prior to morning feeding. Rumen fermentation parameters were measured using ELISA, the content of volatile fatty acids was determined by gas chromatography, and changes in the quantity and structure of the rumen microbiota were analyzed using 16S rRNA amplicon high-throughput sequencing. The results indicated that following the administration of Sijunzi San, the rumen pH remained relatively stable and did not exhibit a significant change (P>0.05). However, the levels of ammonia nitrogen (NH₃-N), microbial protein (MCP), cellulase, and α-amylase experienced a notable increase (P < 0.01), and urease activity also rose (P < 0.05). Conversely, the concentrations of propionic acid and isobutyric acid in the rumen saw a decrease (P < 0.05), whereas the content of n-butyric acid surged (P < 0.01). At the phylum level, Bacteroidota and Firmicutes maintained their predominance as the two dominant bacterial phyla, showing no statistically significant alterations in their relative abundances (P>0.05). However, genus-level analysis revealed distinct shifts in rumen microbiota composition: the relative abundances of Prevotella and Rikenellaceae_RC9_gut_group exhibited a significant increase, while Muribaculaceae showed a marked reduction in abundance. Additionally, the abundance of 10 non-dominant microbiota changed significantly, with 6 beneficial genera significantly upregulated (P < 0.05), including the Eubacterium ruminantium group, Rothia, Bacteroidales RF16 group, Limnohabitans, hgcI clade, and Escherichia Shigella. Four harmful genera were significantly downregulated (P < 0.05), including UCG-001, Clostridium_sensu_stricto_1, Dubosiella, and Parvimonas. Rothia exhibited a positive correlation with α-amylase, propionic acid, and isobutyric acid (P < 0.05); Limnohabitans was positively associated with urease, cellulase, α-amylase, NH₃-N, MCP, propionic acid, and isobutyric acid (P < 0.05), and negatively correlated with pH (P < 0.001); HgcI_clade had a positive correlation with NH₃-N (P < 0.05); Clostridium_sensu_stricto_1 was negatively correlated with pH (P < 0.001); Dubosiella was negatively associated with MCP, NH₃-N, and propionic acid (P < 0.05); and Parvimonas was negatively correlated with MCP and NH₃-N (P < 0.05). The results suggest that the spleen-invigorating effect of Sijunzi San may be closely related to the regulation of rumen microbiota abundance and the enhancement of digestive enzyme activity in the rumen.

Key words: Sijunzi San, rumen fermentation, volatile fatty acids, rumen microbiota, 16S rRNA high-throughput sequencing