Silibinin supplementation alleviates weaning stress in Holstein calves through improved growth, antioxidant response, and gut microbiota

Abstract

This study investigated the effects of dietary silibinin (SLB) supplementation on growth performance, immune status, stress-related plasma biomarkers, and gut microflora composition in Holstein calves. Twenty neonatal female Holstein calves (initial body weight: 42.18 ± 2.65 kg) were assigned to two dietary treatments using a randomized block design, stratified by age and weight. The control group (CON; n = 10) received a basal diet with hydroxypropyl-beta-cyclodextrin (vehicle). The experimental group (n = 10) received the basal diet supplemented with 3.25 mg SLB per kg body weight as a feed additive. During the 61-day experimental period, the calves had ad libitum access to starter feed and water while housed in individual 1.5 m³ calf pens, with rice husks used as bedding, which was replaced every two days. The calves were weaned 56 days of age. Significant improvements in growth performance, including greater final body weight, higher average daily gain, and increased dry matter intake in the pre-weaning phase, were observed with SLB supplementation. Additionally, SLB enhanced fecal scores, antioxidant status and immune function during milk feeding. Plasma analyses revealed reduced levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and lipid peroxidation (malondialdehyde), alongside increased immunoglobulin levels (IgG, IgM) and total superoxide dismutase activity. Furthermore, SLB supplementation improved fecal pH and increased fecal volatile fatty acid concentration both pre- and post-weaning. Microflora analysis indicated minimal shifts in dominant phyla within the hindgut pre- and post-weaning. However, SLB supplementation increased the relative abundance of Bacteroidota while decreasing Firmicutes and Actinobacteriota. At the genus level, SLB increased UCG-005, Lachnoclostridium, Prevotella, and Ruminococcus, but decreased Lactobacillus and Peptoclostridium. Metabolomic analysis of the fecal samples indicated that SLB induced enrichment in pathways for flavonoid biosynthesis, secondary bile acid biosynthesis, and the tricarboxylic acid (TCA) cycle. It increased the production of bile acid metabolites, amino acids, and nicotinic acid, while reducing concentrations of malic acid and citric acid. These results demonstrate that SLB supplementation mitigates stress responses and deleterious effects associated with weaning by enhancing growth performance, antioxidant capacity, gut microflora composition, and intestinal health parameters in dairy calves. This study provides valuable insights into the potential application of SLB in young ruminant nutrition.