Early-life high-dose sodium butyrate supplementation in milk inhibits growth via sterol metabolism in 15-month-old dairy cattle: Insights from gastrointestinal microbiota and host metabolism

ABSTRACT: Sodium butyrate (SB) is a common feed additive used in calf nutrition to support early growth and gastrointestinal health; however, its long-term programming effects remain poorly characterized. This study examined the dose-dependent effects of preweaning SB supplementation in milk on long-term growth, metabolic profiles, and gastrointestinal microbiota in dairy cattle. Eighty Holstein calves were assigned to one of 4 treatments beginning at 2 to 4 d of age: milk supplemented with 0 (CON), 4.4 (LSB), 8.8 (MSB), or 17.6 (HSB) g/d of SB. The same animals were evaluated later as heifers at 15 mo of age for performance, metabolic parameters, and microbial communities. Ruminal fluid, fecal, and plasma samples were collected from 8 animals per group and analyzed via 16S rRNA sequencing (V3–V4 regions) and liquid chromatography-tandem MS–based metabolomics. The HSB group showed a significant reduction in withers height compared with CON, although no significant differences were detected in BW, heart girth, or reproductive measures. Metabolomic and biochemical profiling indicated disrupted sterol metabolism and signs of hepatic stress in HSB heifers, reflected by increased alanine aminotransferase and total bilirubin, alongside decreased total cholesterol and creatine. Ruminal microbiota in the HSB group exhibited reduced diversity, richness, and evenness, accompanied by a decline in beneficial bacteria such as Rikenellaceae_RC9_gut_group. Predicted microbial function indicated inhibited steroid biosynthesis in the rumen. In contrast, the intestinal microbiota composition remained largely unchanged, though steroid degradation function was suppressed. Correlation and network analyses linked these changes, suggesting that early high-dose SB disrupts ruminal microbial ecology, resulting in lasting impairments in host metabolic health and growth. Key biomarkers included Rikenellaceae_RC9_gut_group, steroid biosynthesis, and plasma creatine. Collectively, these results indicate that milk-supplemented high-dose SB in early life leads to long-term inhibitory effects on growth and metabolic homeostasis in dairy heifers, largely mediated through rumen microbiota-driven alterations in sterol metabolism.