Abstract
This study investigated the effects of varying proline supplementation doses in weaned foals. Twenty-eight weaned foals (approximately 5 months of age; body weight: 54.45 ± 11.33 kg; with an equal number of males and females) were randomly assigned to one of four groups-a control group, a low-dose group (20 mg/kg·d), a medium-dose group (40 mg/kg·d), and a high-dose group (60 mg/kg·d)- receiving continuous supplementation for 60 days. Blood samples were collected periodically for the analysis of hormones, antioxidants, immune parameters, and plasma amino acids. Concurrently, fecal 16S rRNA sequencing was performed to assess the microbial community composition. We observed a significant time-dependent interaction between medium-to-high proline supplementation and time. Proline supplementation resulted in dose-dependent increases in foal body weight (p = 0.002), hormone levels (p < 7.49 × 10(-6)), antioxidant capacity (p < 1.56 × 10(-3)), immune function (p < 0.005), and key blood biochemical parameters (p < 0.019). Concurrently, supplementation with medium and high doses of proline significantly reduced the plasma concentrations of amino acids such as proline and arginine (p < 0.05). The medium dose achieved the optimal balance between promoting growth and maintaining high nitrogen utilization efficiency. At the microbial level, medium-dose proline significantly enhanced fecal microbial diversity, particularly enriching characteristic taxa like g_Christensenellaceae_R-7_group. Furthermore, functional inference from PICRUSt2 revealed that medium-dose proline supplementation was associated with a higher microbial potential for amino acid degradation and metabolism, in line with the decreasing plasma concentrations of the corresponding amino acids. This research enhances our understanding of the "host-microbiota interaction" mechanism in weaned foals and provides important theoretical support for mitigating weaning stress and optimizing nutritional strategies.