25-Hydroxyvitamin D3 supplementation improves growth performance and alleviates weaning stress in calves by modulating intestinal microbiota and metabolic pathways.

The purpose of this study was to assess how 25-hydroxyvitamin D3 affected pre-and post weaning calves' growth performance, weaning stress, fecal microbial communities and metabolites, and serum metabolites. A total of 30 healthy Holstein heifers were randomly assigned into a control group (6,100 IU VD3/ day; n = 15) and 25D group (2,100 IU VD3 + 4,000 IU 25(OH)D3/day; n = 15). The findings revealed that 25D considerably improved average daily gain (ADG) and starter intake during the entire experimental period and body weight on d56, 63, while body diagonal length and heart girth on d 63 were greatly increased (p < 0.05). In contrast to the control group, 25D reduced the levels of IL-1β, IL-6, TNF-α, and D-lactate acid (p < 0.05), and the higher levels of 25(OH)D3 and 1,25(OH)2D3 in serum on d 63 (p < 0.05). LEfSe analysis revealed that on day 56, 25D increased the relative abundance of butyrate producing bacteria (e.g., g_Frisingicoccus and g_Lachnospiraceae_NK4A136) and decreased the relative abundance of g_Alistipes associated with pro-inflammation (LDA > 2, p < 0.05). On d 63, 25D further increased the relative abundance of g_Ruminococcus and g_Erysipelotrichaceae_UCG-003. The metabolomic analysis showed that 25D significantly enriched key pathways in the fecal and serum, including alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism, tryptophan metabolism, valine, arginine and proline metabolism, citrate cycle (TCA cycle), pyruvate metabolism, and fructose and mannose metabolism. The downregulation of fecal amino acid metabolites is consistent with the small intestine's utilization of dietary amino acids, which may imply that 25D positively influences intestinal absorption and limits fecal amino acid accumulation. Concurrently, the upregulation of serum fumarate, (S)-malate, and citrate promotes energy metabolism. In conclusion, this study demonstrates that 25D improves calf growth and anti-inflammatory responses by modulating hindgut microbial communities, along with their energy and amino acid metabolic pathways. These findings elucidate the microbial and metabolic mechanisms through which 25D enhances calf health and productivity both before and after weaning, and alleviates weaning stress.